Synthesis and anti-human hepatocellular carcinoma activity of new nitric oxide-releasing glycosyl derivatives of oleanolic acid

Article abstract of DOI:10.1039/b918846k

A series of nitric oxide (NO)-releasing glycosyl derivatives (2-14) of oleanolic acid were synthesized to improve the aqueous solubility and cytotoxicity of the parent compound 1. Derivative 3 exhibited better solubility and strong cytotoxicity against human hepatocellular carcinoma (HCC) in vitro. Furthermore, 3 displayed low acute toxicity to mice and significantly inhibited the growth of HCC tumors in vivo, indicating that 3 may be a promising candidate for the treatment of human HCC.

Full text of DOI:10.1039/b918846k

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and anti-human hepatocellular carcinoma activity of new nitric
oxide-releasing glycosyl derivatives of oleanolic acid†
Zhangjian Huang,a Yihua Zhang,*a Li Zhao,b Yongwang Jing,c Yisheng Lai,a Luyong Zhang,*c Qinglong Guo,b
Shengtao Yuan,c Jianjun Zhang,d Li Chen,a Sixun Penga and Jide Tiane
Received 14th September 2009, Accepted 31st October 2009
First published as an Advance Article on the web 8th December 2009
DOI: 10.1039/b918846k
A series of nitric oxide (NO)-releasing glycosyl derivatives (2–14) of oleanolic acid were synthesized to
improve the aqueous solubility and cytotoxicity of the parent compound 1. Derivative 3 exhibited
better solubility and strong cytotoxicity against human hepatocellular carcinoma (HCC) in vitro.
Furthermore, 3 displayed low acute toxicity to mice and significantly inhibited the growth of HCC
tumors in vivo, indicating that 3 may be a promising candidate for the treatment of human HCC.
as microemulsion and nano-liposomes. Hence, it is unfeasible to
Introduction
make 1 injectable for clinical application. Notably, the glycosyl
Human hepatocellular carcinoma (HCC) is one of the lead-
modification of some drugs can improve their aqueous solubilities
ing causes for mortality in the world.1 Currently, there is no
and cell penetrations, and help in their targeting to specific types
effective chemotherapy for HCC. Therefore, development of new
of cells, enhancing the selectivity and bioactivity through in-
tra/intercellular carbohydrate-protein interaction.4 Glycosylated
drugs have been successfully marketed worldwide.5 In this study,
we synthesized new glycosyl derivatives (2–14) of 1 and examined
their cytotoxicities against HCC cells in vitro. Furthermore, we
evaluated the solubility, NO-releasing ability, in vivo acute toxicity,
and anti-HCC activity of the compound 3. We found that 3
(galactosyl derivative of 1) had better aqueous solubility, produced
high levels of NO, and displayed strong cytotoxicity selectively
against HCC in vitro and in vivo, but little acute toxicity to mice.
We discussed the implication of our novel findings in the future
development of anti-HCC medicines.
therapeutic reagents, particularly injectable drugs, will be of great
significance.
High levels of NO usually are toxic to tumor cells.2 Our
previous studies have demonstrated that furoxan-based NO
releasing derivatives of oleanolic acid (OA), such as the com-
pound 1 (Chart 1), have strong cytotoxicity against HCC in vitro
and significantly inhibit the growth of HCC tumors in vivo.3
However, 1 has a very low aqueous solubility (< 1 mg mL-1 at
pH 6.5–7.8), even with different strategies of formulation, such
aCenter of Drug Discovery, China Pharmaceutical University, Nanjing,
P. R. China 210009. E-mail: zyhtgd@hotmail.com; Fax: +86-25-86635503;
Tel: +86-25-83271186
Results
bJiangsu Key Laboratory of Carcinogenesis and Intervention, China Phar-
maceutical University, Nanjing, P. R. China 210009
Chemistry
cNew Drug Screening Centre, China Pharmaceutical University, Nanjing,
P. R. China 210009. E-mail: lyzhang@cpu.edu.cn; Fax: +86-25-83271500;
Tel: +86-25-83591036
Ten monosaccharide derivatives (eight hexoses, 2–7, 13 and 14,
and two pentoses, 8 and 9) and three disaccharide derivatives
(10–12) of 1 were synthesized by coupling the corresponding
O-acetylated glycosyl bromides to the 28-COOH of 1 and sub-
sequent deacetylation (Schemes 1–4). The glycosyl bromides 15a,
15b, and 15e–15k were obtained by the treatment of saccharides
with Ac2O–CH3COBr–MeOH in one-pot.6 The compound 15c
dDepartment of pharmaceutics, China Pharmaceutical University, Nanjing,
P. R. China 210009
eDepartment of Molecular and Medical Pharmacology, University of
California Los Angeles, Los Angeles, CA, 90095, USA
† Electronic supplementary information (ESI) available: HPLC conditions
chromatograms of 2–14. See DOI: 10.1039/b918846k
Chart 1 The structures of 1, 3, 16 and 17
This journal is
632 | Org. Biomol. Chem., 2010, 8, 632–639
The Royal Society of Chemistry 2010
©

View Article Online
Scheme 1 The synthetic routes of 2–7. Regents and conditions: (i) K2CO3, CTAB, H2O–CH2Cl2, r.t., 48 h for 2, 3, 5–7; or K2CO3, DMF, r.t., 48 h for 4;
(ii) MeONa, MeOH–CH2Cl2, 0–10 ◦C, 30–60 min.
Scheme 2 The synthetic routes of 8 and 9. Regents and conditions: (i) K2CO3, CTAB, H2O–CH2Cl2, r.t., 48 h; (ii) MeONa, MeOH–CH2Cl2, 0–10 ◦C,
30–60 min.
Scheme 3 The synthetic routes of 10–12. Regents and conditions: (i) K2CO3, CTAB, H2O–CH2Cl2, r.t., 48 h; (ii) MeONa, MeOH–CH2Cl2, 0–10 ◦C,
30–60 min.
Scheme 4 The synthetic routes of 13 and 14. Regents and conditions: (i) K2CO3, DMF, r.t., 8 h; (ii) MeONa, MeOH–CH2Cl2, 0–10 ◦C, 20–40 min.
was achieved through the methylation of D-glucuronic acid with
MeONa, and acetylation in Ac2O catalyzed by perchloric acid
and then bromination with 30% hydrobromic acid in acetic acid.7
The compound 15d was derived from peracetylated 2-amino-2-
deoxy-D-glucose in the presence of 30% hydrobromic acid in acetic
acid.8 The compounds 15l and 15m were generated by coupling
1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-D-glucose and galactose
with bromoacetyl bromide, respectively.9 The coupling reaction
of 1 with O-acetylated glycosyl bromides was conducted using
K2CO3 and CTAB in the mixed solvent of H2O and CH2Cl2 (for
2, 3, 5–12), or in the presence of K2CO3 in DMF (for 4, 13 and
14). Without purification, the condensation products were directly
deacylated using MeONa in anhydrous MeOH and CH2Cl2, and
neutralized with acidic ion exchange resin to protect the sensitive
ester bonds in the target molecule. Notably, LiOH in anhydrous
THF was used for the demethylation for the preparation of
4. OA moiety (16) and furoxan moiety (17) were obtained via
condensation of 18 with succinic anhydride and deacylation
(Scheme 5), and by the previous procedure,3 respectively. All
of the compounds were subjected to further purification and
chemical characterization, and their successful syntheses provided
a base for the development of new type of NO-based anti-HCC
drugs.
In vitro assessment of glycosyl derivatives of 1
The cytotoxicity of synthesized compounds against human HCC
SMMC-7721 cells was evaluated by lactate dehydrogenase (LDH)
assays3 using 5-fluorouracil (5-FU) and 1 as positive controls, and
their IC50 are presented in Fig. 1. The IC50 values of 2, 3, 9 and 10
(2.7–8.2 mM) were the same order of magnitude as 1, which was
significantly lower than that of 5-FU (43.5 mM).
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 632–639 | 633
©

View Article Online
Scheme 5 The synthetic route of 16. Regents and conditions: (i) K2CO3, CTAB, H2O–CH2Cl2, r.t., 48 h; (ii) succinic anhydride, DMAP, anhydrous
CHCl3, reflux, 8 h; (iii) MeONa, MeOH–CH2Cl2, 0–10 ◦C, 30 min.
Fig. 2 Cytotoxicity of 3 against HepG2 and LO2 cells. HepG2 and LO2
cells were cultured in medium in the presence of indicated concentrations
of 3 for 24 h. The cytotoxicity of 3 was determined by MTT assay. Data
are means SEM of cytotoxicity (%) from three independent experiments.
Fig. 1 The IC50 of compound 1–14 and 5-FU against human HCC
SMMC-7721 cell.
In comparison with 1, 3 displayed similar and stronger
cytotoxicity against human HepG2, SMMC-7721 and BEL-7402,
respectively, determined by the MTT assay (Table 1).10 Further
analysis revealed that treatment with increased doses of 3, similar
to treatment with vehicle, had no significant effect on the survival
of non-HCC LO2 cells while the same treatment induced majority
of HepG2 cell death (Fig. 2). Therefore, 3 had strong cytotoxicity
selectively to human HCC cells in vitro.
Given that 3 contained both OA (16) and furoxan (17) moieties
(Chart 1), we further characterized the cytotoxicity of 16 and 17
against HCC cells, simultaneously. While 16 had little cytotoxicity
against HCC in our experimental conditions, both 17 and 3
displayed strong cytotoxicity against HepG2 cells (Fig. 3a),
suggesting that NO produced by furoxan and 3 was responsible for
their cytotoxicity. Indeed, treatment with 20 mg ml-1 haemoglobin,
a known NO quencher,11 abolished and significantly reduced the
anti-HCC activities of 17 and 3 (Fig. 3b). Importantly, treatment
with 3 induced high levels of NO production in HepG2 cells, which
was 2- to 5-fold higher than that in LO2 cells (Fig. 2c). Collectively,
our data indicate that the furoxan moiety in 3 promotes high levels
of NO production, which are responsible for its strong cytotoxicity
selectively against HCC cells in vitro.
The solubility of 1 and 3 in aqueous and nonaqueous solutions
was determined, as previously reported.12 We found that 16.2 mg
mL-1 of 3 was soluble in 1,2-propylene glycol and 0.826 mg
mL-1 in 5% macrogol 15 hydroxystearate (solutol HS-15) aqueous
solution, which were obviously higher than 2.23 mg mL-1 and
0.158 mg mL-1 of 1 in corresponding solutions, respectively.
Furthermore, the solubility of 3 in 20% solutol HS15, transcutol
P, 2% tween 80 or labrasol reached at 16.5, 4.6, 2.2 or 1.5 mg
mL-1, respectively. Accordingly, the injectable solution of 3 (5 mg
mL-1 in 5% 1,2-propylene glycol, 5% solutol HS15, 5% anhydrous
ethanol in H2O) was successfully generated for the evaluation of
its biological activity in vivo.
Table 1 Effects of compound 1 and 3 on proliferation in human HCCa
Inhibitory rates
HCC
Compd.
10-4
M
10-5
M
10-6
M
10-7
M
IC50/mM
HepG2
1
3
1
3
1
3
98.0% 85.2% 62.1% 5.3%
99.5% 95.8% 15.7% 1.2%
98.5% 24.2% 10.2% 8.7%
99.6% 98.4% 11.6% 7.9%
1.37
2.13
4.78
1.18
14.5
2.96
SMMC-
7721
BEL -7402
In vivo anti-HCC activity of 3
92.9% 18.0% 5.5%
99.7% 82.6% 7.8%
4.9%
1.1%
To evaluate the safety of 3, groups of mice were injected
intravenously with a single dose of 3 at 150.0, 120.0, 96.0, 76.8,
61.4 mg kg-1 or vehicle control, respectively. The survival of mice
a Data are expressed as mean (%) of three independent experiments.
634 | Org. Biomol. Chem., 2010, 8, 632–639
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 2 The acute toxicity of 3 in mice
Number of dead mice
Dose/mg kg-1
Number of mice
1h
4h
1d
2d
3d
4–14d
Total death
Survival (%) on day 14
150.0
120.0
96.0
76.8
61.4
10
10
10
10
10
10
0
0
0
0
0
0
10
4
1
0
0
—
5
2
0
0
—
0
2
1
0
—
0
1
0
0
—
0
0
0
0
10
9
6
1
0
0
10
40
90
100
100
vehicle
0
0
0
0
0
0
Fig. 3 Determination of relevant components for cytotoxicity. (a) Cytotoxicity of different components. HepG2 cells were treated with 12.5 mM of 3,
16 and 17 and the viability of the cells was determined by MTT assays. (b) Effect of haemoglobin on the cytotoxicity of 3 and 17. HepG2 cells were
pretreated with, or without, 20 mM of haemoglobin for 1 h and then exposed to 12.5 mM of 3 or 17. The viability of the cells was determined by MTT
assays 24 h later. (c) The levels of nitrites/nitrates. HepG2 and LO2 cells were treated with different doses of 3 for 6 h and the intracellular levels of
nitrites/nitrates were determined. Data shown are mean SEM of individual groups from three separated experiments. *P <0.05, **P <0.01 vs. vehicle
control (a), haemoglobin treated (b), or LO2 (c), respectively.
was monitored up to 14 days after injection. Treatment with 3 at
a high dose (120 or 150 mg kg-1) killed almost all mice (Table 2),
and the LD50 value of 3 was 94.1 mg kg-1 for this strain of mice.
In contrast, injection with 3 at 61.4 mg kg-1, like with vehicle
alone, did not cause any abnormality in the mice throughout the
observation period. Therefore, injection with 3 at or below this
dose is supposed to be safe for mice.
To evaluate the in vivo activity of 3, groups of Balb/c nude
mice were inoculated subcutaneously with SMMC7721 cells. After
the establishment of solid tumor, the mice were randomized and
Fig. 4 The effect of 3 on the growth of HCC tumors in vivo. Groups
of Balb/c nude mice were inoculated with SMMC7721 cells. After
establishment of solid tumor, the mice were treated with, or without,
treated with 12.5 or 25 mg kg-1 of 3, positive control 5-FU
(25 mg kg-1) or vehicle alone, respectively. Treatment with vehicle
alone did not change the growth of tumors in vivo because the
growthofHCC tumors in the vehicle-treatedmice was dynamically
similar to that in untreated controls (data not shown). In contrast,
treatment with 3 at 12.5 mg kg-1 significantly inhibited the growth
of HCC tumor (p < 0.01 vs. controls). Treatment with 25 mg kg-1
of 3 enhanced the inhibitory effect on the growth of HCC tumors
(p < 0.01 vs. treatment with 12.5 mg kg-1), and its inhibitory effect
was similar to that of 5-FU treatment (Fig. 4). Importantly, the
tumor weights in the mice treated with 3 at 12.5 or 25 mg kg-1
was 0.48 0.33 g or 0.31 0.13 g, which were significantly lower
than that from the vehicle-treated controls (0.87 0.31 g, p <
0.01). Notably, the mean body weight in the mice treated with 3
was heavier than that of the mice treated with 5-FU at the end
of this experiment. Together, our data demonstrate that the NO-
releasing glycosyl derivative of OA is a potent inhibitor of the
growth of solid HCC tumors and has a better safety in vivo.
indicated reagent and the growth of tumors were measured. Data shown
are mean SEM of tumor volumes for each group of mice (n = 10 per
group). The kinetics of HCC tumor growth in untreated controls were
similar to that solvent controls (data not shown).
that of disaccharide derivatives except for the maltosyl compound.
Among ten monosaccharide derivatives, the glucosyl (2) and galac-
tosyl (3) derivatives displayed the strongest cytotoxicity against
HCC cells. Notably, high levels of glucose transporter proteins and
asialoglycoprotein receptors (ASGPR) are expressed in human
HCC cells4,13 and they may interact with those glucosyl and
galactosyl derivatives by recognization and endocytosis,10 leading
to the efficient entry of those compounds into HCC cells. However,
the precise mechanisms underlying the potent cytotoxicity of
those compounds on HCC cells remain to be further investigated.
Indeed, 3 showed stronger cytotoxicity against different types
of HCC cells (HepG2, SMMC-7721 and BEL-7402), but did
not affect the survival of non-HCC human liver cells in vitro.
The selective cytotoxicity is likely mediated by high levels of
NO produced in HCC cells. Evidently, the furoxan moiety (17)
and 3, but not the OA moiety (16), displayed potent anti-HCC
Discussion and conclusions
Analysis of structure and activity relationship (SAR) revealed that
the monosaccharide derivatives had stronger cytotoxicity than
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 632–639 | 635
©

View Article Online
activity, which was neutralized by NO quencher haemoglobin.
Furthermore, the intracellular levels of NO produced by 3 in HCC
cells were about 2- to 5-fold higher than that in non-HCC LO2
cells. The differential levels of NO produced by 3 between HCC
and non-HCC liver cells may be crucial for its selective anti-HCC
activity.
Interestingly, the anti-HCC activity of 3 was more potent than
that of 17. The higher anti-HCC activity may result from the
more efficient entry of 3 into the cells, the synergistic effect
of NO with OA, and/or higher stability of 3. As expected,
3 had a better aqueous solubility and could be prepared for
intravenous injection. Importantly, intravenous injection with 3
had low acute toxicity in mice. Treatment with 3 at lower doses
significantly inhibited the growth of HCC tumors in nude mice
and its inhibitory effects were dose-dependent. The selective anti-
HCC activity in vitro, lower acute toxicity in mice and highly
inhibitory effect on the growth of HCC tumors in vivo of 3 make it
a promising candidate for preclinical study. Our findings indicate
that NO-releasing glycosyl derivatives of OA may be suitable for
preparing injectable medicine for intervention of human HCC.
resulting residue was purified by column chromatography (E to
MeOH–CH2Cl2 1 : 10 v/v) to give the title compounds (20–36%).
Procedure for the preparation of 4
Compound 1 (852 mg, 1 mmol) and K2CO3 (207 mg, 1.5 mmol)
were dissolved in DMF 10 mL and then reacted with 1.2 mmol
methyl (2,3,4-tri-O-acetyl-a-D-glucopyranosyl bromide)-uronate
in several fractions at RT for 48 h. Subsequently, the mixture was
poured into H2O (150 mL) and the resulting rough compound was
extracted with CH2Cl2 (50 mL¥3). The collected CH2Cl2 layers
were washed, dried and concentrated to obtain oil-like materials,
followed by deacetylation as described above. The generated
residue was dissolved in anhydrous THF 10 mL in the presence of
200 mg LiOH for 24 h and reacted with MeOH (10 mL), followed
by adjusting the pH up to 7.0 with acidic ion exchange resin 001 ¥
7 (732). After filtration and evaporation, the resulting residue was
purified by column chromatography (E to MeOH–CH2Cl2 1 : 8
v/v) to give the title compounds (15%).
General procedure for the preparation of 13 and 14
Compound 1 (852 mg, 1 mmol) and K2CO3 (207 mg, 1.5 mmol)
were dissolved in DMF 10 mL and reacted with 1.2 mmol
corresponding O-acetylated glycosyl bromides in several fractions
at RT for 48 h. Subsequently, the mixture was poured into H2O
(150 mL) and the resulting rough compounds were extracted with
CH2Cl2 (50 mL¥3). The collected CH2Cl2 layers were washed
sequentially with 1 M HCl (80 mL¥3), water and saturated NaCl
solution, dried over sodium sulfate, and concentrated in vacuo to
obtain oil-like materials, which were then deacetylated using the
procedures described above to give the title compounds (19–30%).
Experimental section
General
The compound 1 was prepared as described previously.3 Melting
points were determined using a capillary apparatus (RDCSY-
I). All of the synthesized compounds were purified by column
chromatography (CC) on silica gel 60 (200-300 mesh) or thin-
layer chromatography (TLC) on silica gel 60 F254 plates (250 mm;
Qingdao Ocean Chemical Company, China). Subsequently, they
were routinely analyzed by IR (Shimadzu FTIR-8400S), 1H-NMR
(Bruker ACF-300Q, 500 MHz), and MS (Hewlett-Packard 1100
LC/MSD spectrometer). The purity of the compounds tested,
except for 16 that was analyzed by elemental analysis (Elementar
Vario EL III instrument), was characterized by the HPLC analysis
(LC-10A HPLC system consisting of LC-10ATvp pumps and
SPD-10Avp UV detector) and high resolution mass spectrometry
(Agilent technologies LC/MSD TOF). Individual compounds
with a purity of >95% were used for subsequent experiments (see
the ESI†).
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-
3-yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-glucosyl olea-
nolate (2). The title compound was obtained in 21% yield as
◦
white solid. m.p. 101–103 C. ESI-MS: 1032 [M+NH4]+, 1049
[M+Cl]-; IR (KBr): 3439, 2947, 2879, 1732, 1617, 1553, 1366,
1
1170 cm-1; H NMR (500 MHz, DMSO), d (ppm): 0.75 (s, 3H,
CH3), 0.82 (s, 6H, 2¥CH3), 0.85 (s, 3H, CH3), 0.89 (s, 6H, 2¥CH3),
1.23 (s, 3H, CH3), 2.67 (s, 4H, 2¥COCH2), 2.77~2.88 (m, 1H,
C18–H), 3.65 (m, 1H, H3), 3.78 (m, 1H, H5), 4.09 (m, 1H, H4),
4.18 (brs, 1H, 3a-H), 4.21 (t, 2H, OCH2, J = 6 Hz), 4.43 (m, 1H,
H2), 4.76 (d, 1H, J = 4.5 Hz, H6¢), 4.83 (d, 1H, J = 6.0 Hz, H6),
4.99~5.13 (m, 1H, OCH), 5.26 (brs, 1H, C12–H), 5.49 (d, 1H, J =
8 Hz, H1), 7.61~7.64 (m, 2H, ArH), 7.73~7.76 (m, 1H, ArH), 8.06
(d, 2H, ArH, J = 8.2 Hz). HRMS: calculated 1037.4651, found
1037.4655, PPM error 0.3584.
General procedure for the preparation of 2, 3, 5–12
Compound 1 (852 mg, 1 mmol) was dissolved in the mixture
of 15 mL CH2Cl2 and 20 mL H2O in the presence of K2CO3
(207 mg, 1.5 mmol) and CTAB (91 mg, 0.25 mmol). The mixture
was vigorously stirred and reacted with the corresponding O-
acetylated glycosyl bromides (1.2 mmol) in several fractions at
RT for 48 h. The organic layer was harvested and the remaining
organic solvents in the aqueous layer were extracted with CH2Cl2
(10 mL¥3), followed by drying over sodium sulfate and then
evaporating. The resulting oil-like materials were dissolved in the
mixture of 1 : 1 anhydrous CH2Cl2 and MeOH on ice and its pH
was adjusted to 9.0 with 0.1 M MeONa/MeOH. The deacetylation
was monitored by TLC (1 : 10 v/v MeOH–CH2Cl2) and its pH
was then adjusted to 7.0 with acidic ion exchange resin 001 ¥ 7
(732). After filtration, the filtrate was evaporated in vacuo and the
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-
3-yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-galactosyl olea-
nolate (3). The title compound was obtained in 28% yield as
◦
white solid. m.p. 121–124 C. ESI-MS:1032 [M+NH4]+, 1049
[M+Cl]-; IR (KBr): 3437, 2947, 1733, 1612, 1552, 1367, 1175 cm-1;
1H NMR (500 MHz, DMSO), d (ppm): 0.67 (s, 3H, CH3), 0.74 (s,
6H, 2¥CH3), 0.83 (s, 3H, CH3), 0.87 (s, 6H, 2¥CH3), 1.1 (s, 3H,
CH3), 2.53 (s, 4H, 2¥COCH2), 2.77~2.83 (m, 1H, C18–H), 3.51
(m, 1H, H3), 3.68 (m, 1H, H5), 4.28 (m, 1H, H4), 4.41 (brs, 1H,
3a-H), 4.45 (t, 2H, OCH2, J = 6 Hz), 4.51 (m, 1H, H2), 4.71 (d,
1H, J = 4.5 Hz, H6¢), 4.93 (d, 1H, J = 6.0 Hz, H6), 4.99~5.03 (m,
1H, OCH), 5.16 (brs, 1H, C12–H), 5.21 (d, 1H, J = 8 Hz, H1),
636 | Org. Biomol. Chem., 2010, 8, 632–639
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
7.72~7.75 (m, 2H, ArH), 7.88~7.91 (m, 1H, ArH), 8.01 (d, 2H,
ArH, J = 8 Hz)◦ HRMS: calculated 1037.4651, found 1037.4662,
PPM error 1.0331.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-xylosyl oleanolate
(8). The title compound was obtained in 21% yield as white
◦
solid. m.p. 103–106 C. ESI-MS: 1007 [M+Na]+, 1019 [M+Cl]-;
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-
3-yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-glucuronyl olea-
nolate (4). The title compound was obtained in 15% yield as
white syrup. ESI-MS 1027 [M - H]-; IR (KBr): 3429, 2922, 2851,
1733, 1618, 1553, 1367, 1171 cm-1; 1H NMR (500 MHz, DMSO),
d (ppm): 0.68 (s, 3H, CH3), 0.73 (s, 6H, 2¥CH3), 0.83 (s, 3H, CH3),
0.90 (s, 6H, 2¥CH3), 1.11 (s, 3H, CH3), 2.54 (s, 4H, 2¥COCH2),
2.67–2.93 (m, 1H, C18–H), 3.06–3.10 (m, 2H, H3, H4), 4.28 (m,
1H, 3a-H), 4.39–4.43 (m, 2H, OCH2), 4.91–5.12 (m, 2H, OCH,
H2), 5.12 (d, 2H, J = 5.7 Hz), 5.16 (brs, 1H, C12–H), 5.22 (d, 1H,
J = 8 Hz, H1), 7.71~7.77 (m, 2H, ArH), 7.87~7.92 (m, 1H, ArH),
8.01 (d, 2H, ArH, J = 8 Hz). HRMS: calculated 1028.1884, found
1028.1870, PPM error -1.2621.
IR (KBr): 3424, 2942, 1733, 1618, 1553, 1367, 1170 cm-1; 1H
NMR (500 MHz, DMSO), d (ppm): 0.65 (s, 3H, CH3), 0.74 (s,
6H, 2¥CH3), 0.85 (s, 3H, CH3), 0.91 (s, 6H, 2¥CH3), 1.18 (s,
3H, CH3), 2.54 (s, 4H, 2¥COCH2), 2.62~2.73 (m, 1H, C18–H),
3.68~3.78 (m, 2H, H3, H4), 4.28~4.34 (m, 1H, 3a-H), 4.48~4.55
(m, 2H, OCH2), 4.67~4.81 (m, 2H, H5, H5¢), 4.91~5.00 (m, 1H,
OCH), 5.12~5.22 (m, 1H, H2), 5.28 (brs, 1H, C12–H), 5.32 (d, 1H,
J = 5 Hz, H1), 7.71~7.76 (m, 2H, ArH), 7.89~7.90 (m, 1H, ArH),
7.99~8.02 (m, 2H, ArH). HRMS: calculated 1007.4545 found
1007.4553, PPM error 0.7311.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-arabinosyl olea-
nolate (9). The title compo◦und was obtained in 21% yield
as white solid. m.p. 117–120 C. ESI-MS: 983 [M - H]-, 1019
[M+Cl]-; IR (KBr): 3444, 2947, 1733, 1617, 1552, 1367, 1170 cm-1;
1H NMR (500 MHz, DMSO), d (ppm): 0.68 (s, 3H, CH3), 0.79
(s, 6H, 2¥CH3), 0.81 (s, 3H, CH3), 0.89 (s, 6H, 2¥CH3), 1.08 (s,
3H, CH3), 2.52 (s, 4H, 2¥COCH2), 2.67~2.73 (m, 1H, C18–H),
3.68~3.70 (m, 2H, H3, H4), 4.29~4.30 (m, 1H, 3a-H), 4.41~4.44
(m, 2H, OCH2), 4.57~4.71 (m, 2H, H5, H5¢), 4.98~5.02 (m, 1H,
H2), 4.99~5.08 (m, 1H, OCH), 5.21 (brs, 1H, C12–H), 5.33 (d, 1H,
J = 5 Hz, H1), 7.72~7.76 (m, 2H, ArH), 7.88~7.89 (m, 1H, ArH),
7.99~8.01 (m, 2H, ArH). HRMS: calculated 1007.4545 found
1007.4552, PPM error 0.6318.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-
3-yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-(2-acetylamino-2-
deoxy-glucosyl) oleanolate (5). The title compound was obtained
in 36% yield as white solid. m.p. 142–146 ◦C. ESI-MS 1078
[M+Na]+; IR (KBr): 3424, 2948, 2879, 1734, 1618, 1552, 1370,
1
1170 cm-1; H NMR (500 MHz, DMSO), d (ppm): 0.73 (s, 3H,
CH3), 0.77 (s, 6H, 2¥CH3), 0.89 (s, 3H, CH3), 0.91 (s, 6H, 2¥CH3),
1.21 (s, 3H, CH3), 2.59 (s, 4H, 2¥COCH2), 2.73~2.99 (m, 1H, C18–
H), 3.17 (m, 3H, COCH3), 3.40~3.51 (brs, 1H, NH), 3.64~3.67
(m, 2H, H3, H5), 4.32~4.36 (brs, 1H, 3a-H), 4.40–4.47 (m, 4H,
OCH2, H2, H4), 4.99 (d, 1H, J = 5.5 Hz, H6), 5.03–5.05 (m, 2H,
OCH, H6¢), 5.20 (brs, 1H, C12–H), 5.26 (d, 1H, J = 8.9 Hz, H1),
7.75–7.78 (m, 2H, ArH), 7.91–7.94 (m, 1H, ArH), 8.02–8.04 (m,
2H, ArH). HRMS: calculated 1056.5097, found 1056.5084, PPM
error -1.2613.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-maltosyl oleanolate
(10). The title compound was obtained in 29% yield as white
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-(2-deoxy-glucosyl)
oleanolate (6). The title compound was obtained in 27% yield
as white syrup. ESI-MS: 997 [M - H]-; IR (KBr): 3427, 2945,
syrup. ESI-MS: 1194 [M+NH4 ]+, 1199 [M+Na]+; IR (KBr):
+
3423, 2963, 1733, 1618, 1553, 1368 cm-1;1H NMR (500 MHz,
DMSO), d (ppm): 0.69 (s, 3H, CH3), 0.71 (s, 6H, 2¥CH3), 0.81
(s, 3H, CH3), 0.85 (s, 6H, 2¥CH3), 1.1 (s, 3H, CH3), 2.53 (s, 4H,
2¥COCH2), 2.71~2.83 (m, 1H, C18–H), 3.41~3.43 (m, 1H, H4),
3.61~3.67 (m, 2H, H5¢, H2¢), 3.71~3.78 (m, 1H, H2), 4.13~4.32 (m,
2H, H5, H4¢), 4.38~4.46 (m, 2H, H3, H3¢), 4.54~4.61 (m,1H, H6b’),
4.67~4.83 (m, 1H, H6b), 5.00~5.03 (m, 2H, H6a, H6a’), 5.09~5.21
(m, 4H, OCH, 3a-H, OCH2), 5.23~5.31 (m, 2H, C12–H, H1¢), 5.62
(d, 1H, J = 8 Hz, H1), 7.72~7.75 (m, 2H, ArH), 7.88~7.91 (m, 1H,
ArH), 7.98~8.00 (m, 2H, ArH). HRMS: calculated 1199.5179
found 1199.5173, PPM error -0.5433.
1
2879, 1733, 1617, 1553, 1367, 1170 cm-1; H NMR (500 MHz,
DMSO), d (ppm): 0.57 (s, 3H, CH3), 0.69 (s, 6H, 2¥CH3), 0.77
(s, 3H, CH3), 0.81 (s, 6H, 2¥CH3), 0.98 (s, 3H, CH3), 2.57 (s, 4H,
2¥COCH2), 2.87~2.93 (m, 1H, C18–H), 4.43~3.51 (m, 1H, H4),
3.62–3.79 (3 m, 3H, H5, H6, H6¢), 3.86~4.01 (m, 1H, H3), 4.42
(brs, 1H, 3a-H), 4.43 (t, 2H, OCH2, J = 6 Hz), 4.68~4.98 (m, 1H,
OCH), 5.18 (brs, 1H, C12–H), 5.24 (dd, 1H, H1), 7.73~7.74 (m, 2H,
ArH), 7.88~7.95 (m, 1H, ArH), 7.99~8.07(m, 2H, ArH). HRMS:
calculated 1021.4702, found 1021.4696, PPM error -0.6006.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-(2-deoxy-galatosyl)
oleanolate (7). The title compound was obtained in 21% yield
as white syrup. ESI-MS: 1016 [M+NH4]+; IR (KBr): 3431, 2946,
1732, 1618, 1553, 1367, 1170 cm-1; 1H NMR (500 MHz, DMSO),
d (ppm): 0.61 (s, 3H, CH3), 0.71 (s, 6H, 2¥CH3), 0.82 (s, 3H, CH3),
0.85 (s, 6H, 2¥CH3), 1.2 (s, 3H, CH3), 2.58 (s, 4H, 2¥COCH2),
2.67~2.81 (m, 1H, C18–H), 4.53~3.61 (m, 1H, H4), 3.65~3.82 (m,
3H, H5, H6, H6¢), 3.90~4.0 (m, 1H, H3), 4.41 (brs, 1H, 3a-H), 4.45
(t, 2H, OCH2, J = 6 Hz), 4.78~5.00 (m, 1H, OCH), 5.14 (brs, 1H,
C12–H), 5.21 (dd, 1H, H1), 7.72~7.74 (m, 2H, ArH), 7.88~7.91
(m, 1H, ArH), 7.99~8.01 (m, 2H, ArH). HRMS: calculated
1021.4702, found 1021.4712, PPM error 0.9657.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-cellobiosyl olea-
nolate (11). The title compound was obtained in 23% yield
◦
as white solid. m.p. 126–129 C. ESI-MS: 1175 [M - H]-; IR
1
(KBr): 3422, 2931, 1734, 1617, 1553, 1367, 1170 cm-1; H NMR
(500 MHz, DMSO), d (ppm): 0.65 (s, 3H, CH3), 0.78 (s, 6H,
2¥CH3), 0.89 (s, 3H, CH3), 0.95 (s, 6H, 2¥CH3), 1.13 (s, 3H, CH3),
2.53 (s, 4H, 2¥COCH2), 2.76~2.83 (m, 1H, C18–H), 3.42~3.43
(m, 1H, H4), 3.62~3.67 (m, 2H, H5¢, H2¢), 3.74~3.78 (m, 1H, H2),
4.23~4.33 (m, 2H, H5, H4¢), 4.38~4.46 (m, 2H, H3, H3¢), 4.50~4.61
(m,1H, H6b’), 4.67~4.77 (m, 1H, H6b), 5.00~5.03 (m, 2H, H6a,
H6a’), 5.09~5.18 (m, 4H, OCH, 3a-H, OCH2), 5.27~5.31 (m,
2H, C12–H, H1¢), 5.61 (d, 1H, J = 8 Hz, H1), 7.72~7.75 (m, 2H,
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 632–639 | 637
©

View Article Online
ArH), 7.88~7.91 (m, 1H, ArH), 7.98~8.00 (m, 2H, ArH). HRMS:
calculated 1199.5179 found 1199.5171, PPM error -0.7100.
Cytotoxicity assay in vitro
HepG2 and non-HCC liver LO2 cells at 104 cells/well were in
triplicate cultured in 10% FBS DMEM in 96-well flat-bottom
microplates overnight. The cells were treated with, or without,
different concentrations of each compound for various periods.
During the last 4 h incubation, 30 mL tetrazolium dye (MTT)
solution (5 mg ml-1) was added to each well. The resulting MTT-
formazan crystals were dissolved in 150 mL DMSO, and the
absorbance was spectrophotometrically measured at 570 nm on
an ELISA plate reader. The cell viability was expressed as the
optical density ratio of the treatment to vehicle control. Additional
experiments were performed by pre-treatment of HepG2 cells with,
or without, 20 mM haemoglobin for 1 h and exposure to individual
compounds tested.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-lactosyl oleanolate
(12). The title compound was obtained in 23% yield as white
◦
solid. m.p. 150–153 C. ESI-MS: 1175 [M - H]-; IR (KBr):
3428, 2945, 2879, 1732, 1614, 1553, 1368, 1170 cm-1;1H NMR
(500 MHz, DMSO), d (ppm): 0.65 (s, 3H, CH3), 0.78 (s, 6H,
2¥CH3), 0.89 (s, 3H, CH3), 0.95 s, 6H, 2¥CH3), 1.13 (s, 3H, CH3),
2.53(s, 4H, 2¥COCH2), 2.76~2.83 (m, 1H, C18–H), 3.39~3.43 (m,
1H, H4), 3.59~3.67 (m, 2H, H5¢, H2¢), 3.74~3.80 (m, 1H, H2),
4.13~4.33 (m, 2H, H5, H4¢), 4.36~4.46 (m, 2H, H3, H3¢), 4.50~4.67
(m, 1H, H6b’), 4.67~4.77 (m, 1H, H6b), 4.89~5.02 (m, 2H, H6a,
H6a’), 5.09~5.16 (m, 4H, OCH, 3a-H, OCH2), 5.27~5.32 (m,
2H, C12–H, H1¢), 5.54 (d, 1H, J = 8 Hz, H1), 7.72~7.76 (m, 2H,
ArH), 7.88~7.91 (m, 1H, ArH), 7.98~8.01 (m, 2H, ArH). HRMS:
calculated 1199.5179 found 1199.5178, PPM error -0.1264.
Solubility Studies
The solubility of 1 and 3 was tested, as described previously.10
Briefly, an excess amount of 1 or 3 powder was introduced in
triplicate into 5 ml of each medium (5% propylene glycol, 5%
macrogol 15 hydroxystearate (HS-15), 20% solutol HS15, 20%
transcutol P, 2% tween 80 or 2% labrasol in water) in a small flask
with a glass stopper and shaken at 37.0 ◦C in dark for 3 days.
Aliquots (about 2 ml) of the solution were collected, centrifuged
at 10,000 g for 5 min and suitably diluted. The concentrations of
each compound were determined by HPLC.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-3-
yl]oxy}propoxy)-4-oxobutanoyl]
oxy-12-en-28-[2-N-(2-deoxy-
glucose)-2-oxo]-ethyl oleanolate (13). The title compound was
obtained in 19% yield as white syrup. ESI-MS: 1071 [M - H]-; IR
(KBr): 3424, 2947, 1733, 1619, 1551, 1366, 1170 cm-1; H NMR
1
(500 MHz, DMSO), d (ppm): 0.67 (s, 3H, CH3), 0.77 (s, 6H,
2¥CH3), 0.83 (s, 3H, CH3), 0.89 (s, 6H, 2¥CH3), 1.2 (s, 3H, CH3),
2.58 (s, 4H, 2¥COCH2), 2.79~2.83 (m, 1H, C18–H), 3.52~3.59 (m,
2H, H5, H3), 3.72~3.74 (m, 1H, H4), 3.81 (m, 1H, H2), 4.28 (m,
1H, H4), 4.44~4.45 (m, 4H, 3a-H, OCH2, H6, H6¢), 4.93~4.94 (m,
1H, OCH), 5.00 (brs, 1H, C12–H), 5.21 (m, 1H, H1), 7.72~7.76
(m, 2H, ArH), 7.88~7.91 (m, 1H, ArH), 8.00 (d, 2H, ArH, J =
7.8 Hz). HRMS: calculated 1094.4865 found 1094.4868, PPM
error 0.1901.
Nitrate/nitrite measurement in vitro
NO levels were determined by using a nitrate/nitrite colorimetric
assay kit (Beyotime Institute of Biotechnology), according to the
manufacturers’ instructions. The cells (5 ¥ 106/well) were treated
in triplicate with different concentrations of the compound for
6 h and lyzed. After microfuge ultrafiltration, the contents of
nitrate/nitrite in the lysates were measured. Individual values were
obtained by subtracting the background (diluent treated wells) and
calculated according to the standard curve.
3-[4-(1-Methyl-3-{[5-oxido-4-(phenylsulfonyl)-1,2,5-oxadiazol-
3-yl]oxy}propoxy)-4-oxobutanoyl] oxy-12-en-28-[2-N-(2-deoxy-
galactose)-2-oxo]-ethyl oleanolate (14). The title compound was
obtained in 30% yield as white syrup. ESI-MS: 1071 [M - H]-; IR
1
(KBr): 3426, 2946, 1732, 1618, 1552, 1367, 1170 cm-1; H NMR
(500 MHz, DMSO), d (ppm): 0.68 (s, 3H, CH3), 0.78 (s, 6H,
2¥CH3), 0.84 (s, 3H, CH3), 0.89 (s, 6H, 2¥CH3), 1.1 (s, 3H, CH3),
2.58 (s, 4H, 2¥COCH2), 2.57~2.83 (m, 1H, C18–H), 3.53~3.59 (m,
2H, H5, H3), 3.72~3.74 (m, 1H, H4), 3.92 (m, 1H, H2), 4.34 (m,
1H, H4), 4.34~4.45 (m, 4H, 3a-H, OCH2, H6, H6¢), 4.80~4.94 (m,
1H, OCH), 5.10 (brs, 1H, C12–H), 5.21 (d, 1H, H1, J = 1.5 Hz),
7.72~7.76 (m, 2H, ArH), 7.88~7.91 (m, 1H, ArH), 8.00 (d, 2H,
ArH, J = 7.8 Hz). HRMS: calculated 1094.4865 found 1094.4881,
PPM error 1.3779.
Acute toxicity assay
Seven-week-old male and female KM mice were from Shanghai
SLAC and housed individually in a specific pathogen free facility.
Groups of mice (n = 10 per group) were injected intravenously with
a single dose of 150.0, 120.0, 96.0, 76.8, 61.4 mg kg-1 or vehicle
control, respectively. The mouse death was monitored daily and
recorded up to 14 days after treatment. The experimental protocols
were approved by the Animal Research Protection Committee of
our campus.
3-(3-Carboxy-1-oxopropoxy)-12-en-28-galactosyl
oleanolate
(16). The title compound was obtained from the condensation
of succinyl oleanolic acid with 2, 3, 4, 6-tetra-O-acetyl-a-D-
galactopyranosyl bromide ◦and then deacetylation in 45% yield as
Tumor growth inhibition in vivo
Female Balb/c nude mice at 5–6 weeks old from the Institute
of Laboratory Animal Science, Chinese Academy of Medical
Sciences, were inoculated subcutaneously with 106 SMMC7721
cells. After the formation of solid tumor with a volume of about
100~300 mm3, the tumor-bearing mice were randomized and
treated intravenously with, or without, 12.5, 25 mg kg-1 of 3,
25 mg kg-1 of 5-FU or vehicle alone (0.4 ml) three times per week
for 21 days, respectively. Their body weights were measured and the
growth of tumors was closely monitored, as described previously.3
1
white solid. m.p. 121–123 C. ESI-MS: 717 [M - H]-; H NMR
(500 MHz, DMSO), d (ppm): 0.69 (s, 3H, CH3), 0.80 (s, 6H,
2¥CH3), 0.88–0.89 (s, 9H, 3CH3), 1.02 (s, 3H, CH3), 2.51 (s, 4H,
2¥COCH2), 3.51 (m, 1H, H3), 3.68 (m, 1H, H5), 4.38 (m, 1H, H4),
4.41 (brs, 1H, 3a-H), 4.51 (m, 1H, H2), 4.71 (d, 1H, J = 5.2 Hz,
H6¢), 4.93 (d, 1H, J = 5.8 Hz, H6), 5.16 (brs, 1H, C12–H), 5.21 (d,
1H, J = 8 Hz, H1). Anal. calcd for C40H62O11·2H2O: C, 63.64 H,
8.81; found C, 63.94 H, 8.93.
638 | Org. Biomol. Chem., 2010, 8, 632–639
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
At the end of the experiment, the mice were sacrificed and their
tumors were dissected and measured.
4 Y. Hou, X. Wu, W. Xie, P. G. Braunschweigerb and P. G. Wang,
Tetrahedron Lett., 2001, 42, 825; X. Wu, X. Tang, M. Xian, P. G.
Brauschweigerb and P. G. Wang, Bioorg. Med. Chem., 2002, 10, 2303.
5 C. Ferna´ndez, O. Nieto, J. A. Fontenla, E. Rivas, M. L. de Ceballos
and A Ferna´ndez-Mayoralas, Org. Biomol. Chem., 2003, 1, 767; F.
Bonina, C. Puglia, M. G. Rimoli, D. Melisi, G. Boatto, M. Nieddu, A.
Calignano, G. La Rana and P. De Caprariis, J. Drug Target, 2003, 11,
25.
6 M. Hunsen, D. A. Long, C. R. D’Ardenne and A. L. Smith, Carbohydr.
Res., 2005, 340, 2670.
7 G. N. Bollenback, W. Long, John. D. G. Benjamin and J. A. Lindquist,
J. Am. Chem. Soc., 1955, 77, 3310.
8 V. R. Bouvet and R. N. Ben, J. Org. Chem., 2006, 71, 3619.
9 T. P. Fondy and S. B. Roberts, J. Med. Chem., 1978, 21, 1222.
10 C. S. Cheung, K. K. Chung, J. C. Lui, C. Lau, P. Hon, J. Y. Chan,
K. Fung and S. W. Au, Cancer Lett., 2007, 253, 224.
11 P. Gong, A. I. Cederbaum and N. Nieto, Mol. Pharmacol., 2004, 65,
130.
Acknowledgements
The authors thank “the Research Fund for the Doctoral Program
of Higher Education of China (NO. 20070316007)” and “Scientific
and Technological Major Special Project—Significant Creation of
New Drugs (NO. 2009ZX09103-090) in the Eleventh Five-Year
Plan” for financial support.
References
1 D. M. Parkin, F. Bray, J. Ferlay and P. Pisani, Ca–Cancer J. Clin., 2005,
55, 74.
2 C. M. Pavlos, H. Xu and J. P. Toscano, Free Radical Biol. Med., 2004,
37, 745.
3 L. Chen, Y. Zhang, X. Kong, E. Lan, Z. Huang, S. Peng, D. L. Kaufman
and J. Tian, J. Med. Chem., 2008, 51, 4834.
12 Y. Dong, W. K. Ng, U. Surana and R. B. Tan, Int. J. Pharm., 2008, 356,
130.
13 S. L. Wang, F. B. Yu, T. Y. Jiang, C. S. Sun, T. Y. Wang and J. H. Zhang,
J. Drug Targeting, 2008, 16, 233.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 632–639 | 639
©