Glycosylated diazeniumdiolate-based oleanolic acid derivatives: Synthesis, in vitro and in vivo biological evaluation as anti-human hepatocellular carcinoma agents

Article abstract of DOI:10.1039/c2ob25252j

A series of O²-glycosylated diazeniumdiolate-based derivatives of oleanolic acid (4-19) were synthesized and their anti-human hepatocellular carcinoma (HCC) activities were evaluated. Compound 6 selectively inhibited HCC, but not non-tumor liver cell proliferation. This inhibition was attributed to high levels of nitric oxide (NO) released in HCC cells. Importantly, 6 exhibited low acute toxicity (LD₅₀ = 173.3 mg kg^-1) and potent inhibition of HCC tumor growth in mice (3 mg kg^-1 iv). Furthermore, 6 induced HCC cell apoptosis, which was accompanied by lower mitochondrial membrane potentials and Bcl2 expression, but with higher cytochrome C release, Bax, caspase 3 and 9 expression activities in HCC cells. Collectively, 6 may be a promising candidate drug for the intervention of HCC.

Full text of DOI:10.1039/c2ob25252j

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Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 3882
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PAPER
Glycosylated diazeniumdiolate-based oleanolic acid derivatives: synthesis,
in vitro and in vivo biological evaluation as anti-human hepatocellular
carcinoma agents†
Zhangjian Huang,‡a Junjie Fu,‡a Ling Liu,a Yijun Sun,b Yisheng Lai,a Hui Ji,a Edward E. Knaus,c Jide Tiand
and Yihua Zhang*a
Received 3rd February 2012, Accepted 9th March 2012
DOI: 10.1039/c2ob25252j
A series of O2-glycosylated diazeniumdiolate-based derivatives of oleanolic acid (4–19) were synthesized
and their anti-human hepatocellular carcinoma (HCC) activities were evaluated. Compound 6 selectively
inhibited HCC, but not non-tumor liver cell proliferation. This inhibition was attributed to high levels of
nitric oxide (NO) released in HCC cells. Importantly, 6 exhibited low acute toxicity (LD50 = 173.3 mg
kg−1) and potent inhibition of HCC tumor growth in mice (3 mg kg−1 iv). Furthermore, 6 induced HCC
cell apoptosis, which was accompanied by lower mitochondrial membrane potentials and Bcl2
expression, but with higher cytochrome C release, Bax, caspase 3 and 9 expression activities in HCC
cells. Collectively, 6 may be a promising candidate drug for the intervention of HCC.
molecules of NO from per molecule of diazeniumdiolate) under
Introduction
physiological conditions.8 Given the benefit of releasing a larger
Human hepatocellular carcinoma (HCC) is an aggressive malig-
nancy with a poor prognosis, and is the third leading cause of
cancer death worldwide. Development of new therapeutic agents
to treat HCC is urgently needed. Currently, there is no effective
chemotherapy for HCC.1 Notably, high levels of NO can induce
tumor cell apoptosis2,3 and oleanolic acid (OA) possesses a
unique property of liver-specific metabolism4 and antitumor
activity.5 Our previous studies have demonstrated that 1,2,5-oxa-
diazole-2-oxide (furoxan)-based NO releasing derivatives of OA
(representative compounds 1 and 2, Scheme 1) show strong cyto-
toxicity against HCC in vitro and significantly inhibit the growth
of HCC tumors in vivo.6,7 However, these derivatives have a low
selective cytotoxicity against HCC, which may cause adverse
effects.
amount of NO, the utility of diazeniumdiolates has been exam-
ined in various aspects of antitumor therapy.9,10 Importantly, the
precise O2-protection of diazeniumdiolates may limit the release
of high levels of NO in non-tumor tissue or cells, thereby mini-
mizing unwanted adverse effects. For example, the O2-glycosy-
lated diazeniumdiolate 3 (Scheme 1) requires glycosidase
cleavage to selectively furnish the diazeniumdiolate that then
releases NO to provide significant antitumor activity.11,12 Fur-
thermore, glycosyl derivatives of a NO donor can be recognized
by glucose transporter proteins and asialoglycoprotein receptors
(ASGPR), which are highly expressed in human HCC cells, thus
Diazeniumdiolate is an attractive alternative type of nitric
oxide (NO) donor that is able to release high levels of NO (two
aCenter of Drug Discovery, China Pharmaceutical University, Nanjing,
P.R. China. E-mail: zyhtgd@163.com; Fax: +86–25-83271015;
Tel: +86-25-83271015
bNanjing Keygen Biotech. Co., Nanjing, P.R. China
cFaculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta T6G 2N8, Canada
dDepartment of Molecular and Medical Pharmacology, University of
California-Los Angeles, Los Angeles, California 90095, USA
†Electronic supplementary information (ESI) available: The preparative
procedures and spectrum data for O2-glycosylated diazeniumdiolates
21a–j and 24, HPLC conditions, chromatograms of 21a–j, 4–19 and 24.
See DOI: 10.1039/c2ob25252j
Scheme 1 Chemical structures of furoxan-based OA derivatives 1 and
2 with potent anti-HCC activities, and one representative O2-glycosy-
lated diazeniumdiolate 3.
‡These authors contributed equally to this work.
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Scheme 2 The synthetic route used to prepare compounds 4–19, 23 and 24. Reactions and conditions: (a) NaOMe–MeOH–Et2O, nanometer-sized
TiO2, 25 °C, 48 h. (b) 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, DMF, K2CO3,
−20 °C, 12 h. (c) CF3COOH, CH2Cl2, 3–4 h. (d) DCC, DMAP, anhydrous CH2Cl2, 25 °C, 24 h. (e) NaOMe–MeOH–CH2Cl2, 0–10 °C, 30 min.
enhancing glycosylated NO donor’s selectivity and cyto-
toxicity.13,14 Accordingly, we hypothesized that O2-glycosylated
diazeniumdiolate derivatives of OA would exhibit potent cyto-
toxicity selectively against HCC. Herein, we describe the syn-
using NaOMe in anhydrous MeOH and CH2Cl2 and subsequent
neutralization with acidic ion exchange resin, provided sixteen
target compounds 4–19. Similar deacetylation of 22a and 21b
produced compounds 23 and 24, respectively (Scheme 2).
thesis of
a
group of O2-glycosylated diazeniumdiolate
derivatives of OA 4–19, their ability to release NO, in vitro and
in vivo anti-HCC activities, and possible mechanisms underlying
their actions.
In vitro biological assessment
Anti-HCC activity was determined by incubating human HCC
SMMC-7721 cells with different concentrations of each test
compound for 48 h. The inhibitory effects of individual com-
pounds 4–19 on the proliferation of human HCC SMMC-7721
cells were measured by MTT assay16 using 5-fluorouracil (5-
FU), 1 and 2 as positive controls. Their IC50 values (a dose that
achieved 50% inhibition of cancer cell proliferation) are pre-
sented in Fig. 1a (data for 18 and 19 with a IC50 of >100 μM are
not shown). The potency of 4–6 (IC50 = 3.45–4.21 μM range)
were of the same order of magnitude as for 1 and 2, but signifi-
cantly more potent than that of 5-FU (45.1 μM). Interestingly, 6
displayed more potent inhibition (IC50 = 3.13 μM) against the
Results
Chemistry
The synthetic route used to prepare 4–19 is depicted in
Scheme 2. Three secondary amines possessing a free hydroxyl
group (4-hydroxypiperidine, N-(2-hydroxyethyl)piperazine or N-
methylethanolamine) and 1-Boc-piperazine were allowed to react
with NO gas at atmospheric pressure in the presence of a cataly-
tic amount of nanometer-sized TiO2 at room temperature. These
respective reactions furnished the corresponding diazeniumdioa-
late sodium salts 20a–d, according to our previous protocol.15
O2-Glycosylation of 20a–d with 2,3,4,6-tetra-O-acetyl-α-D-glu-
copyranosyl bromide, or 2,3,4,6-tetra-O-acetyl-α-D-galactopyra-
nosyl bromide, resulted in the respective O2-glycosylated
diazeniumdiolates 21a–h. Deprotection of 21g and 21h with
trifluoroacetic acid afforded amino compounds 21i and 21j.
Coupling of eight diazeniumdiolate compounds 21a–f, 21i or
22j with the OA compounds 22a or 22b,7 respectively, in
the presence of dicyclohexylcarbodiimide (DCC) and 4-N,N-
dimethylaminopyridine (DMAP), followed by direct deacetylation
proliferation of human HepG2 cells than that of 1 and 2 (IC50
15.37 and 5.71 μM, respectively, Fig. 1b).
=
Next, it was found that compound 6 (2–8 μM) inhibited
HepG2 cell proliferation in a concentration-dependent manner,
but 6 had little inhibitory effect on non-tumor liver LO2 cell pro-
liferation in vitro, that is indicative of a selective cytotoxicity
against HCC cells (Fig. 2a). Furthermore, it was found that incu-
bation with 6 (10 μM), or the diazeniumdiolate glycoside 24, but
not the OA compound 23 lacking the NO donor moiety, inhib-
ited the proliferation of HepG2 cells in a time-dependent manner
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Fig. 1 Inhibitory activities of target compounds against HCC cells. (a) IC50 values for compounds 1, 2, 4–17 and 5-FU in SMMC-7721 cells.
(b) Inhibitory activities of compounds 1, 2, 6 and 5-FU against HepG2 cells. Data are expressed as mean or mean SEM value of each compound
from three separate measurements.
Fig. 2 In vitro biological assessments of 6, 23 and 24. (a) Inhibitory effect of 6 on the proliferation of HepG2 and LO2 cells. HepG2 and LO2 cells
were incubated in triplicate with, or without, the indicated concentrations of 6 for 48 h. Cell proliferation was determined using a MTT assay.
(b) Inhibitory activity of 6, 23 and 24 against HepG2 cells. HepG2 cells were incubated in triplicate with, or without, 10 μM of 6, 23 and 24 for 24 or
48 h, and the cell proliferation was determined by MTT assay. Data are means SEM of the inhibition (%) for three independent experiments.
(c) Quantitative measurement of NO production. Compound 6 (50 μM) was cultured in triplicate in vehicle PBS (■, pH 7.4) or PBS containing
β-D-galactosidase (♦, 10 μg mL−1) at 37 °C for 6 h and the levels of NO released were determined using a calorimetric Griess reaction assay.
Percentage NO release was based on a theoretical maximum release of 2 mol of NO from 1 mol of 6. Data are the mean value of NO released from
three independent measurements (n = 3) and intra-group variation from the mean % value was ≤0.2%. (d) The levels of nitrite/nitrate. HepG2 and
LO2 cells were incubated in triplicate with the indicated concentrations of 6 for 6 h and the levels of intracellular nitrite/nitrate were determined using
a calorimetric Griess reaction assay. Data are expressed as mean SEM of individual groups of cells from three separate experiments. (e) Effect of
haemoglobin (H) on the activity of 6 and 24. HepG2 cells were pretreated with, or without, 10 μM haemoglobin for 1 h and then exposed to 10 μM 6
or 24 for 48 h, respectively. Cell proliferation was determined by MTT assays. Data are expressed as mean SEM of individual groups of cells from
three separate experiments. **P < 0.01 vs. 23 (2b), LO2 (2d), or haemoglobin treated (2e), respectively.
(Fig. 2b). Notably, the inhibitory effect of 6 was significantly
more potent than that of 23, and moderately more potent than
that of 24. These data suggest that (i) NO release from the diaze-
niumdiolate is crucial for its anti-HCC effect, and (ii) the syner-
gistic effects of NO release in conjunction with the inhibitory
activity shown by OA are responsible for the high inhibitory
effect of 6 upon HCC cell proliferation. Indeed, treatment of 6
with β-D-galactosidase (pH 7.4, 37 °C, 6 h) significantly
increased the release of NO as expected, relative to that observed
for the vehicle PBS control (Fig. 2c). Apparently, 6 is a relatively
stable compound from which NO release is enhanced by the
enzyme catalyst galactosidase. Credence for this observation is
consistent with the fact that treatment with 6 resulted in higher
levels of NO release in HepG2 cells relative to that in LO2 cells
(Fig. 2d). More importantly, treatment with 10 μM haemoglobin,
a known NO quencher,17 abolished or significantly reduced the
anti-HCC activity of 6 and 24 (Fig. 2e), further demonstrating
the importance of NO release in the anti-HCC activity of 6. Col-
lectively, our data indicate that 6 releases high levels of NO and
that 6 has potent and selective cytotoxicity against HCC cells.
This highly selective cytotoxicity may be attributed to the fact
that 6 is able to effectively enter tumor cells due to its interaction
with glucose transporter proteins and/or ASGPR, which are
highly expressed on human HCC cells, and that high levels of
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Table 1 Acute toxicity of 6 in mice
Table 2 Treatment with 6 inhibits the growth of tumors in vivoa
Mouse mortality
Survival
(%) on
Dose
Tumor volume
(mm3)
Tumor weight
(g)
Dose
Number
Total
Treatment (mg kg−1
)
mg kg−1 of mice
1 h 4 h 2 d 3–14 d mortality day 14
Vehicle
6
10
3
707.2 322.6
353.9 115.6b
0.72 0.22
0.27 0.09c
200
180
162
145.8
10
10
10
10
0
0
0
0
8
6
4
0
0
0
1
0
0
0
0
0
8
6
5
0
20
40
50
a Data shown are the mean SD of tumor volumes and weights for each
group of mice (n = 12 for the vehicle control group and n = 6 for the
group treated with 6). b P < 0.05 relative to the vehicle control group,
determined by the Mann–Whitney U test. c P < 0.01 relative to the
vehicle control group, determined by the Mann–Whitney U test.
100
treatment with 6 (3 mg kg−1 iv dose 3 times per week for 3
weeks) significantly reduced the volumes of HCC tumors. In this
regard, the mean tumor size (volume) and weight in mice treated
with 6 were significantly lower than the tumors in vehicle-treated
control mice (Table 2).
It is relevant that there was no statistical difference in the
average percentage change in body weights post inoculation
between these two groups of mice (data not shown). Together,
these results demonstrate that the O2-glycosylated diazeniumdio-
late-based OA compound 6 is a potent inhibitor of HCC tumors
in mice, and that it has a lower toxicity than the furoxan-based
galactosyl ester 2.7
Fig. 3 Effect of 6 on the growth of HCC tumors in vivo. Groups of
BALB/c nude mice were inoculated with SMMC-7721 cells. When
mice displayed a small solid tumor, the mice were randomly treated with
either 6, or vehicle, three times per week and the growth of tumors were
measured at the indicated time points. Data shown are mean SEM of
tumor volumes from each group of mice (n = 12 for vehicle control
group and n = 6 for the group treated with 6). *P < 0.05 relative to the
vehicle control.
Possible mechanisms underlying the anti-HCC activity exhibited
by compound 6
To determine the effect of
6 on HCC cell apoptosis,
SMMC-7721 cells were incubated with different concentrations
of 6, or OA, for 48 h, and the percentages of apoptotic HCC
cells were determined by FITC-Annexin V/PI staining and flow
cytometry. As shown in Fig. 4, the percentage of annexin V +
apoptotic HCC cells gradually increased for those cells exposed
to increasing concentrations of 6, demonstrating that incubation
with 6 induced HCC cell apoptosis in a concentration-dependent
manner. In contrast, incubations performed using the same con-
centrations of OA did not promote HCC cell apoptosis (data not
shown).
High levels of NO can induce oxidative stress, which can
induce cell apoptosis through caspase-dependent and indepen-
dent pathways, particularly by the intrinsic mitochondrial
pathway.18,19 During the process of apoptosis, apoptotic signals
can result in the loss of mitochondrial membrane potential
(ΔΨM), the release of cytochrome C from mitochondria to cyto-
plasm, a down-regulation of anti-apoptotic protein Bcl2, and an
up-regulation of pro-apoptotic protein Bax, leading to activation
of caspases 3 and 9.20–24 To determine plausible pathways by
which 6 triggered HCC cell apoptosis, SMMC-7721 cells were
incubated with different concentrations (0, 1.5, 3.0, 6.0 μM) of 6
for 48 h prior to staining with the lipophilic mitochondrial probe
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocya-
nine iodide (JC-1). The number of HCC cells with collapsed
mitochondrial membrane potentials in different groups of cells
were determined by flow cytometry analysis (Fig. 5). These
studies showed that incubation with 6 increased the number of
HCC cells with collapsed mitochondrial membrane potentials.
NO are released following glycosidic bond cleavage by galacto-
sidase in HCC cells.
In vivo anti-HCC activity of 6
It is plausible that the O2-glycosylated diazeniumdiolate 6 will
have a good safety profile due to the selective release of high
levels of NO in HCC cells. Accordingly, groups of mice were
injected intravenously with a single dose of 6 at 200, 180, 162
and 145.8 mg kg−1 respectively, to assess its acute toxicity. Sur-
vival of mice was monitored up to 14 days after injection. Only
two mice that had been treated with 6 at the highest dose
(200 mg kg−1) survived (Table 1). In contrast, injection with 6 at
the lowest dose (145.8 mg kg−1) did not cause any death and
abnormality in eating, drinking, body weight, and activity
throughout the observation period. As a result, 6 showed a LD50
of 173.3 mg kg−1 that was much larger than that for 2 (LD50
=
91.4 mg kg−1),7 suggesting that diazeniumdiolate moiety in 6
may have a safe profile superior to its peer furoxan in this strain
of mice using the same assay.
BALB/c nude mice were inoculated subcutaneously with
SMMC-7721 cells to evaluate the in vivo anti-HCC activity of 6.
When the mouse tumor had grown to 5–7 mm in one dimension,
the mice were randomly administered a 3 mg kg−1intravenous
dose of 6, or vehicle as control, three times per week for three
weeks, and tumor growth was monitored by measurement of
tumor volume every three days (Fig. 3). This study showed that
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Further Western blotting analysis revealed that incubation with
6 dramatically increased the relative levels of pro-apoptotic cyto-
chrome C, Bax, caspase 3 and caspase 9 expression, but reduced
the levels of anti-apoptotic Bcl2 expression (Fig. 6a and b) in a
concentration-dependent manner. Subsequent experiments to
examine the levels of activated caspase 3 and 9 using enzymatic
calorimetric assays kits showed that incubation with 6 elevated
the levels of activated caspase 3 and 9 activities in a concen-
tration-dependent manner (Fig. 6c). The increased number of
HCC cells with collapsed mitochondrial membrane potentials,
and an elevated level of caspase 3 and 9 expression, suggest that
6 induces HCC apoptosis through oxidative stress-triggered
mitochondria-related caspase 3 and 9-dependent pathways. We
are currently interested in further studies to investigate how
NO-releasing compounds promote HCC cell apoptosis.
Discussion and conclusions
Structure–activity relationship (SAR) analysis revealed that the
target compounds 16–19 with an amide bond connecting the OA
moiety to the diazeniundiolate showed much less cytotoxicity
compared to compounds 4–15 with an ester linker. It is possible
that these ester compounds may be more easily metabolized to
release NO than the amide compounds. The secondary amine
moiety connected to the N1-atom of the diazeniundiolate moiety
in 4–15 may be a determinant of anti-HCC activity given that
the relative potency order was 4-hydroxypiperidine > N-(2-
hydroxyethyl)piperazine > N-methylethanolamine. Furthermore,
there was no significant difference in anti-HCC activity between
the O2-glucosyldiazeniumdiolate compounds and O2-galactosyl-
diazeniumdiolate compounds (4, 5 vs. 6, 7, 8, 9 vs. 10, 11, 12,
13 vs. 14, 15 and 16, 17 vs. 18, 19, respectively). In this regard,
it is possible that these glucosyl and galactosyl modified deriva-
tives may interact with both glucose transporter proteins and
asialoglycoprotein receptors (ASGPR) that are highly expressed
on human HCC cells, allowing these glycoside derivatives to
enter into the cells by endocytosis.13,14 Support for this belief is
in agreement with the observation that 6 exhibits a potent and
selective cytotoxicity against HCC cells that may be attributed to
its efficient entry into the cells and the high levels of NO selec-
tively released in HCC cells. More importantly, the O2-glyco-
sylated diazeniumdiolate-based OA compound 6 possesses a
low acute toxicity and administration of 6 at a low dose
(3 mg kg−1 iv) significantly inhibited the growth of HCC tumors
in mice relative to that observed for the less potent furoxan-
based OA derivative 2.7 One plausible explanation for this obser-
vation is that the diazeniumdiolate moiety in 6 can selectively
release more NO in tumor cells relative to furoxan-based OA
derivative 2. Furthermore, it was found that incubation with 6
induced apoptosis in HCC cells, mitochondrial membrane poten-
tials and anti-apoptotic Bcl2 expression were lowered. In con-
trast, increased cytochrome C release and pro-apoptotic Bax,
Fig. 4 Compound 6 induces SMMC-7721 cell apoptosis in vitro.
SMMC-7721 cells were incubated with the indicated concentrations of 6
or OA for 48 h and the cells were stained with FITC-Annexin V and PI,
followed by flow cytometry analysis. (a) Flow cytometry analysis.
(b) Quantitative analysis of apoptotic cells. Data are representative flow
cytometry charts that are illustrated as mean SEM of the percentages
of apoptotic cells from three independent experiments.
Fig. 5 Effect of 6 on the mitochondrial membrane potentials of SMMC-7721 cells. SMMC-7721 cells were incubated with the indicated concen-
trations of 6 for 48 h prior to staining with JC-1. The percentage of cells with healthy or collapsed mitochondrial membrane potentials was determined
by flow cytometry analysis. Data for representative charts are illustrated as mean SEM for the percentages of cells in different phases from three sep-
arate experiments.
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Fig. 6 Effect of 6 on the expression of apoptosis-related proteins in SMMC-7721 cells. (a) The expression of Bax, Bcl2, caspase 3 and 9, cyto-C,
and β-actin was examined by Western blot analysis. SMMC-7721 cells were incubated with, or without, 6 at the indicated concentrations for 48 h and
the levels of protein expression were detected using specific antibodies. Data shown are representative images of each protein for three separate exper-
iments. (b) Quantitative analysis: the relative levels of each protein compared to control β-actin were determined by densimetric scanning. Data are
expressed as means SEM from three separate experiments. (c) The activities of caspase 3 and 9: SMMC-7721 cells were incubated with, or without,
6 at the indicated concentrations for 48 h and the cells were harvested. The contents of activated caspase 3 or 9 in the cell lysates was analyzed using a
colorimetric assay. Data are expressed as means SEM for the relative activities of each group of cells from three separate experiments where the
activity of control cells without 6 treatment was designated as 1. *P < 0.05 vs. the vehicle control. **P < 0.01 vs. the vehicle control.
caspase 3 and 9 expression was observed in HCC cells. Ap-
parently, 6 induces HCC cell apoptosis via oxidative stress-
triggered mitochondria-related caspase-dependent pathways. The
selective in vitro anti-HCC activity, low acute toxicity and high
inhibition of HCC tumor growth in mice treated with 6 make it a
promising candidate drug for the intervention of HCC. It is
expected that the mechanistic and biological studies described in
this investigation will facilitate the design of new therapeutic
agents for the clinical intervention of HCC disease.
(P R China), and all animal experimental protocols were
approved and guided by the Animal Research Protection Com-
mittee of China Pharmaceutical University, Nanjing, China.
General procedure for the preparation of compounds 4–19
A mixture of an O2-glycosylated diazeniumdiolate (1 mmol),
3-oxy-monosuccinate-oleanolic acid-28-β-D-(2,3,4,6-tetra-O-acetyl)-
glycosyl ester (1 mmol), DCC (309 mg, 1.5 mmol) and DMAP
(183 mg, 1.5 mmol) in anhydrous CH2Cl2 (10 mL) was stirred at
room temperature for 12 h at which time the TLC indicated that
the starting materials were totally consumed. After filtration, the
filtrate was concentrated and the residue was dissolved in the
1 : 1 mixture of anhydrous CH2Cl2 and MeOH at ice-bath temp-
erature and MeONa in methanol solution (30%, 0.923 mL) was
added drop wise. The deacetylation procedure was monitored by
TLC and upon completion the reaction mixture was neutralized
with acidic ion exchange resin 001× 7 (732). After filtration, the
filtrate was evaporated in vacuo and the resulting residue was
purified by column chromatography (MeOH–CH2Cl2 1 : 10 v/v)
to furnish the respective title product.
Experimental section
General
Melting points were determined on a Mel-TEMP II melting
point apparatus which was uncorrected. Infrared (IR) spectra
(KBr) were recorded using a Nicolet Impact 410 instrument
(KBr pellet). 1H NMR spectra were recorded with a Bruker
Avance 300 MHz spectrometer at 300 °K, using TMS as an
internal standard. MS spectra were recorded on a Shimadzu
GC-MS 2010 (EI) or a Mariner Mass Spectrum (ESI). Analytical
and preparative TLC were performed on silica gel
(200–300 mesh) GF/UV 254 plates and the chromatograms were
visualized under UV light at 254 and 365 nm. All solvents were
reagent grade and, when necessary, were purified and dried by
standards methods. The purity of all compounds tested was
characterized by the HPLC analysis (LC-10A HPLC system con-
sisting 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†).
O2-β-D-Glucopyranosyl1-{4-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy] piperidin-1-yl}diazen-1-ium-
1,2-diolate (4). The title product was obtained in 33% yield as a
white powder; mp 103–105 °C; IR (KBr, cm−1): 3425, 2946,
1
1733, 1641, 1462, 1257, 1164, 1073, 1010; H NMR (DMSO-
d6, 300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 3H, CH3), 0.88
(s, 6H, 2 × CH3), 1.02–1.04 (m, 3H, CH3), 1.09 (s, 3H, CH3),
4.01–4.06 (m, 1H, H3), 4.34–4.35 (m, 1H, H3), 4.41–4.42
(m, 2H, 2 × H5), 4.58–4.64 (m, 1H, 3α-H), 4.79–4.87 (m, 1H,
OCH), 4.89–4.91 (m, 1H, H4), 4.94–4.96 (m, 1H, H2),
5.02–5.04 (m, 2H, H6′ and H2), 5.12–5.14 (m, 1H, H6′),
5.16–5.18 (m, 2H, 2 × H6), 5.22–5.25 (m, 1H, H1), 5.41–5.43
(m, 1H, H1). ESI-MS: 1022 [M − H]−; 1058 [M + Cl]−; 1041
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide,2,3,4,6-
tetra-O-acetyl-α-D-galactopyranosyl bromide, 22a, 22b and 23
were synthesized, as previously described.11 Sodium diazenium-
diolates 20a–d were prepared according to the method described
previously.15 All animal experiments were performed in accord-
ance with the Animals (Scientific Procedures) Act 2009
[M
+
NH4]+; HRMS: calculated for C51H81N3O18Na
[M + Na]+:1046.5407, found: 1046.5405, PPM error −0.19.
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O2-β-D-Glucopyranosyl 1-{4-[(12-en-28-β-D-galactopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy] piperidin-1-yl}diazen-1-ium-
1,2-diolate (5). The title product was obtained in 28% yield as a
white powder; mp 107–109 °C; IR (KBr, cm−1): 3435, 2948,
1736, 1643, 1459, 1230, 1165, 1075; 1H NMR (DMSO-d6,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.88 (s,
6H, 2 × CH3), 1.10 (s, 3H, CH3), 1.24 (s, 3H, CH3), 2.57 (s, 4H,
2 × COCH2), 2.73–2.77 (m, 1H, C18–H), 3.17–3.23 (m, 4H, 2 ×
NCH2), 3.39–3.42 (m, 4H, 2 × OCH2), 3.47–3.50 (m, 2H, 2 ×
H3), 3.65–3.70 (m, 2H, 2 × H5), 4.38–4.41 (m, 2H, 2 × H4),
4.49–4.51 (m, 1H, H2), 4.55–4.60 (m, 1H, 3α-H), 4.74–4.77 (m,
1H, H2), 4.80–4.87 (m, 1H, OCH), 4.89–4.96 (m, 1H, H6′), 4.98
(d, J = 6.0 Hz, 1H, H6′), 5.03 (d, J = 5.4 Hz, 1H, H6), 5.13–5.16
(m, 2H, H6 and C12–H), 5.21 (d, J = 7.8 Hz, 1H, H1), 5.40–5.42
(m, 1H, H1); ESI-MS: 1022 [M − H]−; 1058 [M + Cl]−; HRMS:
calculated forC51H81N3O18Na [M + Na]+:1046.5407, found:
1046.5388, PPM error −1.82.
2 × H3), 3.60–3.65 (m, 2H, 2 × H5), 4.02–4.04 (m, 1H, H4),
4.11 (t, J = 5.4 Hz, 2H, NCH2), 4.40–4.42 (m, 2H, OCH2), 4.60
(t, J = 5.7 Hz, 1H, 3α-H), 4.88–4.91 (m, 1H, H2), 4.98 (d, J =
4.8 Hz, 1H, H2), 5.01–5.03 (m, 2H, C12–H and H6′), 5.12 (d, J =
4.2 Hz, 1H, H6′), 5.16 (d, J = 5.4 Hz, 2H, 2 × H6), 5.24 (d, J =
7.8 Hz, 1H, H1), 5.41 (d, J = 4.8 Hz, 1H, H1); ESI-MS: 1053 [M
+ H]+; HRMS: calculated for C52H85N4O18 [M + H]+:
1053.5853, found: 1053.5858, PPM error 0.47.
O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-galactopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy]ethylpiperazin-1-yl}diazen-1-
ium-1,2-diolate (9). The title compound was obtained in 26%
yield as a white powder; mp 142–145 °C; IR (KBr, cm−1): 3420,
2946, 1735, 1631, 1463, 1161, 1147; 1H NMR (DMSO-d6,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.88
(s, 6H, 2 × CH3), 1.10 (s, 3H, CH3), 1.12–1.24 (m, 3H, CH3),
2.55–2.60 (m, 4H, 2 × COCH2), 2.73–3.79 (m, 1H, C18–H),
3.17–3.21 (m, 4H, 2 × NCH2), 3.40–3.43 (m, 4H, 2 × NCH2),
3.45–3.51 (m, 2H, 2 × H3), 3.63–3.72 (m, 3H, H5 and NCH2),
4.02–4.04 (m, 1H, H5), 4.11 (t, J = 5.7 Hz, 2H, NCH2),
4.38–4.47 (m, 1H, H4), 4.50–4.51 (m, 1H, H4), 4.55–4.62
(m, 2H, H2 and 3α-H), 4.77 (d, J = 5.1 Hz, 1H, H2), 4.90 (d, J =
7.8 Hz, 1H, H6′), 4.97 (d, J = 5.7 Hz, 1H, H6′), 5.03 (d, J = 5.1
Hz, 1H, H6), 5.11–5.13 (m, 1H, H6), 5.17 (brs, 1H, C12–H), 5.21
(d, J = 7.5 Hz, 1H, H1), 5.41 (d, J = 4.5 Hz, 1H, H1); ESI-MS:
1053 [M + H]+; 1087 [M + Cl]−; HRMS: calculated for
C52H85N4O18 [M + H]+:1053.5853, found: 1053.5844, PPM
error −0.85.
O2-β-D-Galactopyranosyl 1-{4-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy] piperidin-1-yl}diazen-1-ium-
1,2-diolate (6). The title compound was obtained in 29% yield
as a white powder; mp 106–108 °C; IR (KBr, cm−1): 3421,
2946, 1734, 1654, 1457, 1162, 1074; 1H NMR (CDCl3,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.88 (s,
9H, 3 × CH3), 1.09 (s, 3H, CH3), 2.57 (s, 4H, 2 × COCH2),
3.38–3.52 (m, 6H, 2 × H5 and 2 × NCH2), 3.60–3.63 (m, 1H,
H3), 3.67 (s, 1H, H3), 4.39–4.42 (m, 2H, H4 and 3α-H), 4.51 (d,
1H, J = 3.5 Hz, H4), 4.63 (brs, 1H, H2), 4.82–4.87 (m, 3H, OCH
and 2 × H6′), 4.93 (s, 1H, H2), 4.99 (brs, 1H, C12–H), 5.13
(d, J = 5.0 Hz, 1H, H6), 5.17 (brs, 1H, H6), 5.22–5.25 (m, 2H,
2 × H1); ESI-MS: 1041 [M + NH4]+; HRMS: calculated for
C51H81N3O18Na [M + Na]+:1046.5407, found: 1046.5416, PPM
error 0.86.
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy]ethylpiperazin-1-yl}diazen-1-
ium-1,2-diolate (10). The title compound was obtained in 39%
yield as a white powder; mp 144–146 °C. IR (KBr, cm−1): 3421,
1
1734, 1645, 1464, 1231, 1160; H NMR (CDCl3, 300 MHz): δ
O2-β-D-Galactopyranosyl 1-{4-[(12-en-28-β-D-galactopyrano-
0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.86–0.88 (m, 6H,
2 × CH3), 1.09 (s, 3H, CH3), 1.23 (s, 3H, CH3), 2.55–2.58
(m, 4H, 2 × COCH2), 3.08–3.17 (m, 4H, 2 × NCH2), 3.41–3.54
(m, 4H, 2 × NCH2), 3.61–3.66 (m, 2H, 2 × H3), 4.09–4.11
(m, 2H, 2 × H5), 4.37–4.44 (m, 2H, 2 × H4), 4.54 (d, J = 4.5 Hz,
1H, H2), 4.64–4.67 (m, 1H, 3α-H), 4.85 (d, J = 8.4 Hz, 1H, H2),
4.90 (d, J = 5.7 Hz, 1H, H6′), 4.96 (d, J = 4.8 Hz, 1H, H6′), 5.02
(d, J = 4.8 Hz, 1H, H6), 5.16–5.18 (m, 2H, H1 and H6),
5.22–5.26 (m, 1H, H1); ESI-MS: 1053 [M + H]+; 1051
[M − H]−;HRMS: calculated for C52H85N4O18 [M + H]+:
1053.5853, found: 1053.5852, PPM error −0.09.
syl-oleanolate-3-yl-oxy)-succinyl-oxy]piperidin-1-yl}
diazen-1-
ium-1,2-diolate (7). The title compound was obtained in 25%
yield as a white powder; mp 104–106 °C; IR (KBr, cm−1): 3421,
1
2947, 1732, 1642, 1468, 1162, 1147, 1072. H NMR (CDCl3,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3),
0.87–0.88 (m, 9H, 3 × CH3), 1.09 (s, 3H, CH3), 2.56 (s, 4H, 2 ×
COCH2), 2.73–2.76 (m, 1H, C18–H), 3.66–3.68 (m, 2H, 2 ×
H3), 4.38–4.41 (m, 1H, H4), 4.47 (d, J = 4.2 Hz, 1H, H2),
4.49–4.53 (m, 2H, H2 and H4), 4.62 (t, J = 5.3 Hz, 1H, 3α-H),
4.73 (d, J = 5.3 Hz, 1H, H6′), 4.82–4.86 (m, 3H, H6, H6′ and
OCH), 4.94 (d, J = 5.7 Hz, 1H, H6), 5.16 (brs, 1H, C12–H),
5.20–5.22 (m, 2H, 2 × H1); ESI-MS: 1022 [M − H]−, 1058
[M + Cl]−; 1041 [M + NH4]+; HRMS: calculated for
C51H81N3O18Na [M + Na]+:1046.5407, found: 1046.5399, PPM
error −0.76.
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-galactopyrano-
syl-oleanolate-3-yl-oxy)-succinyl-oxy]ethylpiperazine-1-yl}diazen-
1-ium-1,2-diolate (11). The title compound was obtained in 21%
yield as a white powder; mp 141–143 °C; IR (KBr, cm−1): 3421,
2923, 1735, 1643, 1464, 1231, 1147, 1069, 1003; 1H NMR
(CDCl3, 300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 2H, 2 × CH3),
0.88 (s, 6H, 2 × CH3), 1.10 (s, 3H, CH3), 1.24 (s, 3H, CH3),
2.55 (s, 4H, 2 × COCH2), 3.49–3.53 (m, 2H, 2 × H3), 3.66–3.67
(m, 2H, 2 × H5), 4.11 (t, J = 4.8 Hz, 2H, OCH2), 4.39–4.41
(m, 1H, H4), 4.49–4.54 (m, 3H, 2 × H2 and H4), 4.62–4.67
(m, 1H, 3α-H), 4.75–4.78 (m, 1H, H6′), 4.84–4.90 (m, 2H, H6′
and H6), 4.97 (d, J = 5.4 Hz, 1H, H6), 5.16–5.25 (m, 3H, C12–H
O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy] ethylpiperazin-1-yl}diazen-1-
ium-1,2-diolate (8). The title compound was obtained in 23%
yield as a white powder; mp 140–143 °C; IR (KBr, cm−1): 3423,
2946, 1735, 1641, 1462, 1161; 1H NMR (DMSO-d6, 300 MHz):
δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.87–0.88 (m, 6H,
2 × CH3), 1.10 (s, 3H, CH3), 1.24 (s, 3H, CH3), 2.55 (s, 4H,
2 × COCH2), 2.72–2.78 (m, 1H, C18–H), 3.12–3.14 (m, 4H,
2 × NCH2), 3.32 (m, 4H, 2 × NCH2), 3.45–3.47 (m, 2H,
and
2
×
H1); HRMS: calculated for C52H85N4O18
[M + H]+:1053.5853, found: 1053.5852, PPM error −0.09.
3888 | Org. Biomol. Chem., 2012, 10, 3882–3891
This journal is © The Royal Society of Chemistry 2012

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O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy] ethyl-N-methylamino}diazen-
1-ium-1,2-diolate (12). The title compound was obtained in 19%
yield as a white powder; mp 118–120 °C; IR (KBr, cm−1): 3421,
4.60–4.65 (m, 1H, 3α-H), 4.76–4.77 (m, 1H, H6′), 4.83–4.86 (m,
1H, H6), 4.88–4.92 (m, 1H, H6′), 4.98 (d, J = 5.4 Hz, 1H, H6),
5.16 (brs, 1H, C12–H), 5.20–5.24 (m, 2H, 2 × H1); ESI-MS:
1016 [M + NH4]+; 1021 [M + Na]+; HRMS: calculated for
C49H79N3O18Na [M + Na]+:1020.5251, found: 1020.5243, PPM
error −0.78.
1
2921, 1733, 1642, 1464, 1199, 1068, 1020; H NMR (CDCl3,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.89
(s, 6H, 2 × CH3), 1.10 (s, 3H, CH3), 1.24 (s, 3H, CH3), 2.55
(s, 4H, 2 × COCH2), 2.73–2.76 (m, 1H, C18–H), 2.99 (s, 3H,
NCH3), 3.54–3.69 (m, 4H, 2 × H3 and 2 × H5), 4.15–4.18
(m, 2H, NCH2), 4.35–4.48 (m, 3H, OCH2 and H4), 4.54–4.57
(m, 1H, 3α-H), 4.89 (d, J = 7.2 Hz, 1H, H4), 4.95 (d, J = 4.8 Hz,
1H, H2), 5.01–5.02 (m, 2H, H2 and H6′), 5.11–5.13 (m, 1H, H6′),
5.15–5.17 (m, 2H, 2 × H6), 5.24 (d, J = 8.1 Hz, 1H, H1),
5.38 (d, J = 4.2 Hz, 1H, H1); ESI-MS: 1016 [M + NH4]+;
O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl] piperazin-1-yl}diazen-1-ium-1,2-
diolate (16). The title compound was obtained in 45% yield as a
white powder; mp 181–183 °C; IR (KBr, cm−1): 3423, 2945,
1729, 1640, 1466, 1387, 1231, 1177; 1H NMR (CDCl3,
300 MHz): δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3),
0.87–0.88 (m, 6H, 2 × CH3), 1.10 (s, 3H, CH3), 1.18–1.24
(m, 3H, CH3), 2.73–2.75 (m, 1H, C18–H), 3.56–3.65 (m, 4H, 2
× H5 and 2 × H3), 4.33–4.44 (m, 1H, H4), 4.40–4.44 (m, 2H, 2
× H4), 4.54 (d, J = 4.8 Hz, 1H, H2), 4.76 (d, J = 5.4 Hz, 1H,
H2), 4.62–4.67 (m, 1H, 3α-H), 4.86–4.92 (m, 2H, H2 and H6′),
4.94 (d, J = 4.5 Hz, 1H, H6′), 5.01 (d, J = 4.5 Hz, 1H, H6),
5.16–5.18 (m, 2H, H6 and C12–H), 5.22–5.27 (m, 2H, 2 × H1);
997 [M
−
H]−; HRMS: calculated for C49H79N3O18Na
[M + Na]+:1020.5251, found: 1020.5252, PPM error 0.10.
O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-galactopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy]ethyl-N-methylamino}diazen-1-
ium-1,2-diolate (13). The title compound was obtained in 20%
yield as a white powder; mp 115–117 °C; IR (KBr, cm−1): 3421,
2945, 1734, 1695, 1464, 1160, 1073; 1H NMR (CDCl3,
300 MHz): δ 0.69 (s, 3H, CH3), 0.81 (s, 2H, 2 × CH3), 0.86
(s, 6H, 2 × CH3), 0.95 (s, 3H, CH3), 1.11 (s, 3H, CH3), 1.25
(s, 3H, CH3), 2.72–2.79 (m, 1H, C18–H), 2.99 (s, 3H, NCH3),
3.62–3.68 (m, 2H, 2 × H3), 4.14–4.19 (m, 2H, 2 × H5),
4.38–4.40 (m, 1H, H4), 4.50–4.58 (m, 3H, 2 × H2 and H4),
4.61–4.66 (m, 1H, 3α-H), 4.74–4.79 (m, 1H, H6′), 4.81–4.85 (m,
1H, H6), 4.86–4.90 (m, 1H, H6′), 4.97–5.03 (m, 1H, H6),
5.15–5.27 (m, 3H, C12–H and 2 × H1); ESI-MS: 1016 [M +
NH4]+; 1021 [M + Na]+; 997 [M − H]−; 1033 [M + Cl]−;
HRMS: calculated for C49H79N3O18Na [M + Na]+:1020.5251,
found: 1020.5203, PPM error −4.70.
ESI-MS: 1043 [M
+
Cl]−; HRMS: calculated for
C50H80N4O17Na [M + Na]+:1031.5411, found: 1031.5412, PPM
error 0.10.
O2-β-D-Glucopyranosyl 1-{N-[(12-en-28-β-D-galactopyranosyl-
oleanolate-3-yl-oxy)-succinyl] piperazin-1-yl}diazen-1-ium-1,2-
diolate (17). The title compound was obtained in 40% yield as a
white powder; mp 182–184 °C; IR (KBr, cm−1): 3431, 1730,
1
1640, 1466, 1231, 1177, 1146; H NMR (CDCl3, 300 MHz): δ
0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.88 (s, 9H, 3 × CH3),
1.10 (s, 3H, CH3), 2.60–2.61 (m, 4H, 2 × COCH2), 2.75–2.81
(m, 1H, C18–H), 3.22–3.26 (m, 4H, 2 × NCH2), 3.30–3.40 (m,
4H, 2 × NCH2), 3.45–3.47 (m, 2H, 2 × H3), 3.60–3.63 (m, 2H,
2 × H5), 4.35–4.42 (m, 1H, H4), 4.50 (d, J = 4.2 Hz, 1H, H4),
4.55–4.60 (m, 2H, 3α-H and H2), 4.77 (d, J = 5.4 Hz, 1H, H2),
4.90–4.93 (m, 1H, H6′), 4.98 (d, J = 6.0 Hz, 1H, H6′), 5.03
(d, J = 5.4 Hz, 1H, H6), 5.13–5.16 (m, 2H, H6 and C12–H), 5.21
(d, J = 7.8 Hz, 1H, H1), 5.42 (d, J = 4.8 Hz, 1H, H1); ESI-MS:
1043 [M + Cl]−; HRMS: calculated for C50H80N4O17Na
[M + Na]+:1031.5411, found: 1031.5417, PPM error 0.58.
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl-oxy]ethyl-N-methylamino}diazen-
1-ium-1,2-diolate (14). The title compound was obtained in 20%
yield as a white powder; mp 119–121 °C; IR (KBr, cm−1): 3423,
1
2927, 1737, 1640, 1464, 1262, 1161, 1072; H NMR (CDCl3,
300 MHz): δ 0.68 (s, 3H, CH3), 0.80 (s, 2H, 2 × CH3), 0.88
(s, 6H, 2 × CH3), 0.94 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.23
(s, 3H, CH3), 2.72–2.76 (m, 1H, C18–H), 2.97 (s, 3H, NCH3),
3.66 (m, 2H, 2 × H3), 4.14–4.16 (m, 2H, 2 × H5), 4.38–4.41
(m, 1H, H4), 4.51–4.54 (m, 3H, 2 × H2 and H4), 4.61–4.64
(m, 1H, 3α-H), 4.76–4.77 (m, 1H, H6′), 4.83–4.86 (m, 1H, H6),
4.88–4.91 (m, 1H, H6′), 4.96–5.02 (m, 1H, H6), 5.16–5.29
(m, 3H, C12–H and 2 × H1); ESI-MS: 1033 [M + Cl]−; HRMS:
calculated for C49H79N3O18Na [M + Na]+:1020.5251, found:
1020.5242, PPM error −0.88.
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-glucopyranosyl-
oleanolate-3-yl-oxy)-succinyl] piperazin-1-yl}diazen-1-ium-1,2-
diolate (18). The title compound was obtained in 52% yield as a
white powder; mp 184–186 °C; IR (KBr, cm−1): 3423, 2921,
1
2851, 1731, 1640, 1465, 1231; H NMR (CDCl3, 300 MHz): δ
0.69 (s, 3H, CH3), 0.80 (s, 2H, 2 × CH3), 0.86–0.88 (m, 6H, 2 ×
CH3), 1.09 (s, 3H, CH3), 1.23 (s, 3H, CH3), 2.72–2.75 (m, 1H,
C18–H), 3.61–3.64 (m, 4H, 2 × H3 and 2 × H5), 4.37–4.44
(m, 1H, 2 × H4), 4.54 (d, J = 4.2 Hz, 1H, H2), 4.65 (m, 1H,
3α-H), 4.85–4.92 (m, 2H, C12–H and H6′), 5.01 (d, J = 4.5 Hz,
1H, H6′), 5.16–5.18 (m, 2H, 2 × H6), 5.22–5.27 (m, 2H, 2 × H1);
ESI-MS: 1026 [M + NH4]+; 1043 [M + Cl]−; HRMS: calculated
for C50H80N4O17Na [M + Na]+:1031.5411, found: 1031.5416,
PPM error 0.48.
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-galactopyrano-
syl-oleanolate-3-yl-oxy)-succinyl-oxy]ethyl-N-methylamino}diazen-
1-ium-1,2-diolate (15). The title compound was obtained in 34%
yield as a white powder; mp 115–117 °C; IR (KBr, cm−1): 3449,
1
3328, 1734, 1627, 1576, 1438, 1369, 1312; H NMR (CDCl3,
300 MHz): δ 0.68 (s, 3H, CH3), 0.80 (s, 2H, 2 × CH3), 0.88
(s, 6H, 2 × CH3), 0.94 (s, 3H, CH3), 1.09 (s, 3H, CH3), 1.23
(s, 3H, CH3), 2.72–2.76 (m, 1H, C18–H), 2.97 (s, 3H, NCH3),
3.65–3.69 (m, 2H, 2 × H3), 4.14–4.16 (m, 2H, 2 × H5),
4.38–4.41 (m, 1H, H4), 4.51–4.54 (m, 3H, 2 × H2 and H4),
O2-β-D-Galactopyranosyl 1-{N-[(12-en-28-β-D-galactopyrano-
syl-oleanolate-3-yl-oxy)-succinyl] piperazin-1-yl}diazen-1-ium-
1,2-diolate (19). The title compound was obtained in 39% yield
as a white powder; mp 185–188 °C; IR (KBr, cm−1): 3431,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 3882–3891 | 3889

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1
2945, 1731, 1640, 1466, 1231; H NMR (CDCl3, 300 MHz):
Medical Sciences, were inoculated subcutaneously with 106
SMMC-7721 cells. After the formation of a solid tumor with a
volume of about 100–300 mm3, the tumor-bearing mice were
randomized and treated intravenously with 3 mg kg−1 of 6, or
vehicle alone (0.4 mL) three times per week for 21 days, respect-
ively. The growth of implanted tumors in mice and their body
weights were measured every three days, as described pre-
viously.6 At the end of the experiment, the mice were sacrificed,
their tumors were dissected, and the tumor size and weight were
measured.
δ 0.69 (s, 3H, CH3), 0.80 (s, 6H, 2 × CH3), 0.88 (s, 9H, 3 × CH3),
1.10 (s, 3H, CH3), 1.24 (s, 3H, CH3), 2.73–2.77 (m, 1H,
C18–H), 3.40–3.47 (m, 4H, 2 × NCH2), 3.49–3.55 (m, 4H,
2 × NCH2), 3.61–3.68 (m, 4H, 2 × H3 and 2 × H5), 4.36–4.41
(m, 1H, H4), 4.51 (d, J = 4.5 Hz, 1H, H4), 4.54–4.58 (m, 2H,
2 × H2), 4.63–4.68 (m, 1H, 3α-H), 4.77 (d, J = 5.1 Hz, 1H, H6′),
4.86–4.89 (m, 1H, H6′), 4.90–4.92 (m, 1H, H6), 4.98 (d, J = 5.4
Hz, 1H, H6), 5.16 (brs, 1H, C12–H), 5.21 (d, J = 7.5 Hz, 1H,
H1), 5.27 (d, J = 5.7 Hz, 1H, H1); ESI-MS: 1007 [M − H]−;
1043 [M + Cl]−; HRMS: calculated for C50H80N4O17Na
[M + Na]+:1031.5411, found: 1031.5404, PPM error −0.68.
Flow cytometry assay of cell apoptosis
SMMC-7721 cells were cultured overnight and incubated in
triplicate with the test compound (1.5, 3.0 and 6.0 μM) or
vehicle for 48 h. The cells were harvested, and stained with
FITC-Annexin V and PI at room temperature for 15 min. The
percentage of apoptotic cells were determined by flow cytometry
analysis.
MTT assay
Human HCC SMMC-7721, HepG2 or non-tumor liver LO2
cells at 104 cells per well were cultured in 10% FBS DMEM in
96-well flat-bottom microplates overnight. The cells were incu-
bated in triplicate with, or without, different concentrations of
each test compound for various periods. During the last 4 h incu-
bation, 30 μL of tetrazolium dye (MTT) solution (5 mg mL−1
)
Cell mitochondrial membrane potential assay
was added to each well. The resulting MTT-formazan crystals
were dissolved in 150 μL DMSO, and absorbance was measured
spectrophotometrically at 570 nm using an ELISA plate reader.
The inhibition induced by each test compound at the indicated
concentrations was expressed as a percentage ((1 − the optical
density ratio of the treatment to vehicle control) × 100%).
Additional experiments were performed by pre-treatment of
HepG2 cells with, or without, 20 mM hemoglobin for 1 h and
exposure to individual compounds tested.
SMMC-7721 cells were cultured overnight and incubated in tri-
plicate with the test compound (1.5, 3.0 and 6.0 μM) or vehicle
for 48 h. The cells were stained with JC-1 dye, according to the
manufacturer’s instruction (Keygen, KGA601). The percentage
of cells with healthy or collapsed mitochondrial membrane
potentials was monitored by flow cytometry analysis (Ex =
488 nm; Em = 530 nm).
Western blotting assays
Nitrate–nitrite measurement in vitro
SMCC-7721 cells were incubated in triplicate with different
doses of 6 for 48 h, the cells were harvested, the cells were lyzed
using lysis buffer, and solution was centrifuged. After the
protein concentrations were determined, individual cell lysates
(50 μg per lane) were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (10% gel, SDS-PAGE) and
transferred onto nitrocellulose membranes. After being blocked
with 5% fat-free milk, the target proteins in the membranes
were probed with monoclonal anti-Bax (KGA714), anti-Bcl2
The levels of NO were determined using a nitrate–nitrite colori-
metric assay kit (Beyotime Institute of Biotechnology), accord-
ing to the manufacturers’ instructions. The cells (5 × 106 per
well) were treated in triplicate with different concentrations of
the test compound for 6 h and lyzed. After microfuge ultrafiltra-
tion, the contents of nitrate–nitrite in the lysates were measured.
Individual values were obtained by subtracting the background
(vehicle treated wells) and the amount of nitrite–nitrate was cal-
culated, according to the standard curve.
(KGA715), anti-caspase
3
(KGA717), anti-caspase
9
(KGA720), anti-cyto C (KGA723) and anti-β-actin antibodies
(KGA731, KeyGEN Biotech, Nanjing, China), respectively. The
bound antibodies were detected by horse radish peroxidase
(HRP)-conjugated second antibodies and visualized using the
enhanced chemiluminescent reagent. The relative levels of
each signaling event to control β-actin were determined by
densimetric scanning.
Acute toxicity
Seven-week-old male and female KM mice, purchased from the
Shanghai Laboratory Animal Center (Shanghai, China), were
housed in a pathogen free facility. Groups of mice (n = 10 per
group) were injected intravenously with a single dose of 200,
180, 162, 145.8 mg kg−1 or vehicle control, respectively. Mouse
survival was monitored up to 14 days after treatment. The exper-
imental protocols were evaluated and approved by the Ethics
Committee of China Pharmaceutical University.
Assay of caspase 3, and 9 activity
The activities of caspase 3 and 9 were determined by enzymatic
assays using caspase 3 and 9 colorimetric assay kits, according
to the manufacturers’ instructions (Keygen, KGA202, KGA402).
Briefly, SMCC-7721 cells (3–5 × 106 per well) were incubated
in duplicate with compound 6 (at 0, 1.5, 3.0 and 6.0 μM) for
48 h. The cells were harvested and lyzed in 50 μl of lysis buffer
In vivo tumor growth inhibition
Female BALB/c nude mice 5–6 weeks of age, purchased from
the Institute of Laboratory Animal Science, Chinese Academy of
3890 | Org. Biomol. Chem., 2012, 10, 3882–3891
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