Synthesis and evaluation of the anticancer activity of albiziabioside A and its analogues as apoptosis inducers against human melanoma cells

Article abstract of DOI:10.1039/c4ob00874j

We have efficiently synthesized albiziabioside A (1) together with its six disaccharide analogues through a linear synthesis, and evaluated their cytotoxicity against six different skin cancer cells. All of the analogues showed weak cytotoxicity, with the

Full text of DOI:10.1039/c4ob00874j

Organic &
Biomolecular Chemistry
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Synthesis and evaluation of the anticancer activity
of albiziabioside A and its analogues as apoptosis
inducers against human melanoma cells†
Cite this: Org. Biomol. Chem., 2014,
12, 5928
Gaofei Wei, Shuai Wang, Shanshan Cui, Jia Guo, Yongxiang Liu, Yang Liu* and
Maosheng Cheng*
We have efficiently synthesized albiziabioside A (1) together with its six disaccharide analogues through a
linear synthesis, and evaluated their cytotoxicity against six different skin cancer cells. All of the analogues
showed weak cytotoxicity, with the exception of compound 1, which exhibited strong cytotoxicity against
A375 cells. Albiziabioside A can induce cell cycle arrest in both the S and G2/M phases. Moreover, albizia-
bioside A can induce A375 cell apoptosis via mitochondrial pathways involving a caspase cascade. These
results provide for the first time a basic mechanism for the anticancer activity of 1.
Received 29th April 2014,
Accepted 11th June 2014
DOI: 10.1039/c4ob00874j
www.rsc.org/obc
cytotoxicity towards different tumor cell lines.12–16 We were
recently attracted to a natural oleanane triterpenoid saponin
Introduction
Skin cancer is one of the most extensively diagnosed types of (which we have named albiziabioside A) that was originally iso-
cancer in Caucasians and in those living in equatorial lati- lated from the aerial parts of Albizia inundata.17 Albiziabioside
tudes, and currently accounts for approximately 40% of all A (1) exhibits potent cytotoxic activity against various skin
diagnosed cancers.1,2 Common skin cancers are classified as cancer cell lines, including the murine melanoma cell line
either melanoma or non-melanoma, and malignant melanoma B16F10, the human melanoma cell line SKMEL28, and the
is the predominant type resulting in death. Multiple reports head and neck squamous carcinoma cell line MDA1986 and
have demonstrated that chronic exposure to ultraviolet radi- the JMAR cell line.17
ation is one of the main causes of skin cancer. In addition,
Despite these reports, there has been no previous study
much effort has been focused on developing new approaches on the mechanism of the cytotoxicity of 1. Decreased rates of
to treat skin cancer.3,4 However, the successful treatment apoptosis in skin cancer cells are closely associated with a
of skin cancer remains very challenging because of the un- high risk of tumorigenesis.18–20 Thus, induction of apoptosis
pleasant side effects of the therapies and drug resistance. in skin cancer cells is considered highly useful in therapy and
Therefore, continuing the search for and development of for the prevention of cancer. In the present study, we report
alternative drugs are clinically necessary.
the synthesis and biological activity of 1 and its analogues as
Oleanane triterpenoids, which are synthesized by squalene apoptosis inducers against A375 human melanoma cells.
cyclization in many terrestrial plants and in low marine organ-
isms,5 represent an important family of natural products
widely used in Asian medicine.6 Studies have shown that
oleanane triterpenoids can be used to treat most cancers
through the regulation of a wide range of dysfunctional
pathways, including apoptosis, the cell cycle, growth factor
signaling, inflammation, drug resistance, metastasis, and
angiogenesis.7–11 Recent studies have shown that natural sapo-
Results and discussion
nins chaining glucosamine are presenting strong in vitro
Synthesis of compound 1 and its analogues 12a–12f
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of
The sugar donors used for glycosylation in this study (Fig. 1)
were perbenzoylated sugar trichloroacetimidate derivatives
Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, P. R. China. E-mail: y.liu@syphy.edu.cn,
except for 13,14 which are stable and easily monitored by
mscheng@syphy.edu.cn; Tel: +86 24 23986413, +86 24 23986419
absorption at 254 nm than those acetylated trichloroacetimi-
date derivatives.
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00874j
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Table 1 Effects of 1 and 12a–12f on the growth of six skin cancer cell
lines in vitro
IC50 (μM)
A431
B16BL6
OCM1
SKMEL24
HT144
A375
1
12.19
19.84
16.84
16.04
22.01
>50
7.34
19.16
16.98
18.12
24.42
>50
9.64
18.1
19.76
16.21
27.21
40.93
6.27
8.02
19.74
18.1
18.72
15.61
44.31
9.48
8.27
24.1
27.42
25.89
26.37
>50
5.47
14.69
15.91
18.64
20.82
21.53
26.09
35.28
12a
12b
12c
12d
12e
12f
5-FU
8.45
26.18
8.28
28.14
26.05
>50
49.02
38.30
9 in dry THF at 0 °C, and the key intermediate 10 was obtained
after stirring.23 Condensation of 10 with various sugar donors
(14–20), as described for the preparation of 3, generated 11
and 11a–11f, which were subjected to hydrogenation with H2
over Pd/C. Finally debenzoylation and deacetylation with a cata-
lytic amount of NaOMe in DCM–MeOH furnished the target
compound albiziabioside A (1) and its analogues 12a–12f.
Fig. 1 Chemical structures of glycosyl trichloroacetimidates 13–20.
Cytotoxic effects in six skin cancer lines
To evaluate the effects of 1 and 12a–12f on the growth of skin
cancer cells, the growth inhibitory potential was evaluated
using an MTT assay. Compared with derivatives 12a–12f, 1
exhibited the strongest growth inhibition in each cell line,
suggesting that the 6-OH arabinose of acetylglucosamine
is more effective than other monosaccharides, as shown in
Table 1. In addition, albiziabioside A exhibited outstanding
growth inhibition of all tested skin cancer cell lines with IC50
values below 15 μM. The lowest IC50 values for 1, 5.47 μM, was
obtained in A375 cells compared to other cell lines. Concen-
trations of 1 up to 10 μM primarily resulted in cell-cycle arrest;
concentrations exceeding 20 μM of 1 showed cytotoxicity, and
floating dead cells were observed (Fig. 2A). As shown in
Fig. 2B, cells treated with 1 exhibited morphological changes
and showed distinctly rounded shapes and detachment in a
dose-dependent manner compared to the vehicle-treated
control cells. This result inspired us to investigate the mechan-
ism of action of 1 against A375.
Scheme 1 Synthesis of compound 1 and its analogues 12a–12f.
Albiziabioside A (1) induces S-phase and G2/M-phase cell cycle
arrest
As shown in Scheme 1, the naturally abundant oleanolic
acid was used as the starting material to afford 3 in good yield
after treatment with BnBr and TBAF in dry CH2Cl2. Glycosyl-
ation of 3 using the trichloroacetimidate 13 in dry CH2Cl2 in
the presence of TMSOTf at 0 °C gave 4 in excellent yield. The
addition of zinc to a solution of 4 in acetic acid resulted in the
selective removal of the Troc group.21 This was followed by
acetylation with acetic anhydride in pyridine to afford 6.
Deacetylation of 6 with MeONa in CH2Cl2–MeOH furnished 7.
Then, the primary 6-OH group on 7 was selectively protected
with a TBS group using TBSCl and imidazole,22 and acetyl pro-
tection of the 3,4-OH groups was performed to provide 9.
A catalytic amount of TBAF in acetic acid was slowly added to
Cell proliferation and growth generally occur as a progression
through three phases (G0/G1, S, and G2/M) of the cell cycle,
which is an important chemotherapeutic target. To investigate
whether growth inhibition induced by 1 was associated with
regulation of the cell cycle, the cell cycle distribution in the
presence of 1 was analyzed by flow cytometry. As shown in
Fig. 3, following treatment with 1 at 0.31, 1.25 and 5.00 μM for
72 h, there was an increase in the S fraction of cells (37.87%,
42.42% and 43.32%, compared to 37.28% in untreated cells)
and in G2/M (9.62%, 10.42% and 16.10%, compared to
4.59% in untreated cells), and a decrease in the G0/G1 phases
(52.52%, 47.16% and 41.58%, compared to 58.12% in
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Fig. 2 Inhibitory effects of 1 on A375 cell growth. (A) A375 cells were treated with 1 (80, 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 and 0.16 μM) for 72 h. (B)
Morphological changes by 1 with 1 (20, 10, 5, 2.5 and 1.25 μM) in A375 cells.
untreated cells). These data show that 1 can induce blockade
of the cell cycle at both the S and G2/M phases.
Mitochondria are necessary mediators that play a vital role
in the execution of both intrinsic and extrinsic apoptotic
pathways. To estimate the role of mitochondria in 1-induced
A375 cell apoptosis, we explored mitochondrial membrane
potential (MMP) changes by flow cytometric analysis after JC-1
staining experiments. After treatment with 0 to 5.0 μM of com-
pound 1, MMP marked losing was observed relative to controls
(Fig. 5). So we can conclude that 1 induced the membrane
permeability of mitochondria to increase.
Subsequent experiments were undertaken to analyze
changes in the expression of apoptosis-related proteins that
result from 1; these effects were assayed using Western blot-
ting. The release of mitochondrial cytochrome c into the cyto-
plasm is a crucial event preceding the activation of a caspase
cascade involving caspase-9, -3, and -8.24 As shown in Fig. 6,
Albiziabioside A (1) causes A375 cell death by apoptosis
Apoptosis is programmed cell death and involves a highly
organized physiological mechanism to destroy injured or
abnormal cells in multicellular organisms. To investigate
whether A375 cell death induced by 1 is mediated by apopto-
sis, flow cytometric analysis after Annexin V-FITC antibody
binding and propidium iodide staining of A375 cells was
performed after treatment with 1 at various concentrations.
Following treatments with vehicle or 1 (0.31, 1.25 or 5 μM) for
72 h, the proportions of cells displaying early stage (A+P−)/late
stage (A+P+) apoptosis were 3.18%/0.10%, 9.55%/2.83%,
24.58%/10.31% and 31.2/22.90%, respectively (Fig. 4). These
results show that 1 can induce A375 cell apoptosis, especially
early apoptosis, efficiently and in a dose-dependent manner.
the levels of cytochrome
c released from mitochondria
increased from 0.11 to 0.83 in a dose-dependent manner in
the presence of 1. This increase was correlated with an
increase in the levels of cytochrome c. Furthermore caspase-3,
caspase-9 and caspase-8 activation was also detected, and the
levels increased from 0.11 to 0.6, 0.13 to 0.53 and 0.34 to 0.74,
respectively. Compound 1 also significantly down-regulates the
expression of Bcl-2, which prevents mitochondrial protein
release.25 In contrast, the level of the Bax anti-apoptotic
Albiziabioside A (1) induces A375 cell apoptosis via
mitochondrial pathways involving a caspase cascade
In general, apoptosis occurs via either an intrinsic or an
extrinsic pathway, which involves the induction of pro-
apoptotic proteins or the suppression of anti-apoptotic
proteins, respectively.
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Fig. 3 Effects of 1 on A375 cell cycle progression. A375 cells were
treated with 1 at various concentrations (0, 0.31, 1.25 and 5.00 μM) for
72 h, then harvested, stained with PI, and analyzed by flow cytometry.
Fig. 4 Apoptosis in A375 cells induced by 1. A375 cells were treated
with 1 at various concentrations (0, 0.31, 1.25 and 5.0 μM) for 72 h and
then harvested, stained with Annexin V/PI, and analyzed by flow
cytometry.
protein increased from 0.36 to 0.93 in a dose-dependent
manner.
To evaluate whether 1-mediated apoptosis is caspase-
dependent, A375 cells were pretreated with Z-VAD-fmk (50 μM),
a potent caspase inhibitor, and then treated with 1 (0.31, 1.25,
and 5.00 μM). As shown in Fig. 7, Z-VAD-fmk effectively and
significantly inhibited 1-mediated apoptosis.
To investigate the expression levels of apoptosis-
related genes (Bcl-2, Bax and caspase-3) in the mitochondrial
pathways, we used real-time PCR experiments. As shown in
Fig. 8, we demonstrated that the Bcl-2 gene level was down-
regulated, and this finding was in concordance with the
decrease in the Bcl-2 protein levels from a previous experi-
ment. Similarly, Bax and caspase-3 gene levels were up-
regulated.
All of the results obtained (MMP, apoptosis-related
proteins, Z-VAD-fmk on 1-induced apoptosis, and apoptosis-
related genes) suggested that 1 induces apoptosis via a
mitochondrial pathway involving a caspase cascade.
Conclusion
Fig. 5 Effect of 1 on the A375 cells mitochondrial membrane potential.
A375 cells were treated with 1 at various concentrations (0, 0.31, 1.25
and 5.0 μM) for 72 h and then harvested, stained with JC-1, and analyzed
We have efficiently synthesized albiziabioside A (1) through a
linear synthesis and also obtained 6 new disaccharide ana- by flow cytometry.
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Fig. 8 Effect of 1 on expression of apoptosis-related genes. A375 cells
were treated with 1 at various concentrations (0, 0.31, 1.25 and 5.00 μM)
for 72 h. Then Bcl-2, Bax, and caspase-3 were analyzed using real-time
PCR experiments. GAPDH was used as an internal control.
Experimental
Synthesis
Materials and equipment. Reagents were used without further
purification unless otherwise specified. Solvents were dried
and redistilled prior to use in the usual way. Analytical TLC
was performed using silica gel HF254. Preparative column
chromatography was performed with silica gel H. Melting
points were obtained on a Büchi melting point B-540 appar-
Fig. 6 Effect of 1 on expression of apoptosis-related proteins. A375
cells were treated with 1 at various concentrations (0, 0.31, 1.25 and
5.00 μM) for 72 h. Then cyto-c, Bcl-2, Bax, caspase-9, caspase-8,
caspase-3 and β-actin were analyzed using Western blotting. β-Actin
was used as an internal control.
1
atus. H and 13C NMR spectra were recorded on a Bruker ARX
600 MHz spectrometer. HR-MS were obtained on a Bruker
micrOTOF_Q spectrometer.
28-O-Benzyl-3-O-[2-(2,2,2-trichloroethoxylcarbonylamino)-
3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl] oleanolic ester
(4). A mixture of 3 (2.00 g, 3.66 mmol) and trichloroacetimi-
date 13 (2.74 g, 4.39 mmol) in dry CH2Cl2 (50 mL) was stirred
at rt for 30 min. Then the mixture was cooled to 0 °C and
TMSOTf (0.07 mL, 0.36 mmol) was added. After stirring at this
temperature for 3 h, Et3N was added. The resulting mixture
was filtered. The filtrates were concentrated to give a residue,
which was purified by a silica gel column chromatography
(6 : 1 petroleum ether–EtOAc) to afford 4 (3.45 g, 93.6%) as a
white foam. Mp 105.3–106.9 °C; 1H NMR (600 MHz, CDCl3)
δ 7.38–7.27 (m, 5H), 5.31–5.23 (m, 2H), 5.18–4.99 (m, 1H), 4.72
(d, J = 12.0 Hz, 1H), 4.64 (d, J = 10.2 Hz, 2H), 4.26 (dd, J = 12.1,
5.5 Hz, 1H), 4.10 (dd, J = 12.1, 2.5 Hz, 1H), 3.71–3.63 (m, 2H),
3.10 (dd, J = 11.8, 4.5 Hz, 1H), 2.90 (dd, J = 13.9, 4.5 Hz, 1H),
2.07 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.11 (s, 3H), 0.91 (s, 3H),
0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.76 (s, 3H), 0.59 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 177.6, 170.9, 170.8, 169.6, 154.0,
143.9, 136.6, 128.6, 128.1, 128.1, 122.6, 103.2, 95.3, 91.0, 74.8,
72.1, 71.7, 69.1, 66.1, 62.5, 57.0, 55.6, 47.8, 46.9, 46.1, 41.8,
Fig. 7 Effect of Z-VAD-fmk on 1-induced apoptosis. A375 cells were
preincubated with or without 20 μM Z-VAD-fmk for 3 h, then were
treated with indicated concentrations of 1, and stained with Annexin V/
PI. Cells were analyzed by flow cytometry to evaluate apoptosis.
logues. In vitro biological evaluation demonstrated the strong 41.5, 39.5, 39.1, 38.6, 36.9, 34.0, 33.3, 32.8, 32.5, 30.9, 28.2,
cytotoxicity of 1 in skin cancer cells. 1 induces cell cycle arrest 27.8, 26.0, 25.9, 23.8, 23.6, 23.2, 22.8, 20.9, 20.8, 18.3, 17.0,
at both the S and G2/M phases. Moreover, 1 induces A375 cell 16.6, 15.4; HRMS(ESI): calcd for [M + Na]+ C52H72Cl3NNaO12:
apoptosis via mitochondrial pathways involving a caspase 1030.4018, found 1030.4012.
cascade. These findings support the potential of 1 as a promis-
28-O-Benzyl-3-O-[2-amino-3,4,6-tri-O-acetyl-2-deoxy-β-D-gluco-
ing natural product for the treatment of skin cancer, although pyranosyl] oleanolic ester (5). To a mixture of 4 (3.00 g,
additional studies are necessary to further study this 2.98 mmol) in acetic acid (50 mL), freshly activated zinc
compound.
powder (8.76 g, 13.40 mmol) was added. After it was stirred for
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4 h, the reaction mixture was filtered and diluted with CH2Cl2. 18.4, 17.0, 16.9, 15.5; HRMS(ESI): calcd for [M + Na]+
Then the organic layer was washed with saturated aqueous C45H67NNaO8: 772.4764, found 772.4759.
NaHCO3 and brine, dried over anhydrous Na2SO4, filtered, and
28-O-Benzyl-3-O-[2-acetamido-6-O-tert-butyldimethylsilyl-2-
concentrated to afford 5 (2.39 g, 96.5%) as a white foam. Mp deoxy-β-D-glucopyranosyl] oleanolic ester (8). To a solution of
1
75.6–87.3 °C; H NMR (600 MHz, CDCl3) δ 7.40 −7.28 (m, 5H), 7 (1.54 g, 2.05 mmol) in CH2Cl2 (30 mL), imidazole (1.40 g,
5.29 (dd, J = 6.8, 2.0 Hz, 1H), 5.02 (m, 1H), 4.30 (d, J = 7.9 Hz, 20.50 mmol) and TBSCl (1.54 g, 10.25 mmol) were added.
1H), 4.26 (dd, J = 12.0, 5.6 Hz, 1H), 4.08 (dd, J = 12.0, 2.1 Hz, After it was stirred for 10 min, the mixture was filtered. Then
1H), 3.70–3.62 (m, 1H), 3.16 (dd, J = 11.8, 4.4 Hz, 1H), 2.98 (dd, the filtrate was concentrated and purified by a silica gel
J = 9.7, 8.1 Hz, 1H), 2.90 (dd, J = 13.7, 4.0 Hz, 1H), 2.06 (s, 3H), column chromatography (50 : 1, CH2Cl2–MeOH) to afford 8
1
2.06 (s, 3H), 2.01 (s, 3H), 1.11 (s, 3H), 1.00 (s, 3H), 0.91 (s, 3H), (1.36 g, 76.7%) as a white solid. Mp 153.2–155.7 °C; H NMR
0.89 (s, 3H), 0.88 (s, 3H), 0.82 (s, 3H), 0.60 (s, 3H); 13C NMR (600 MHz, CDCl3) δ 7.39–7.28 (m, 5H), 5.28 (s, 1H), 5.06 (dd,
(150 MHz, CDCl3) δ 177.3, 170.6, 169.7, 143.7, 136.3, 128.3, J = 32.2, 12.6 Hz, 1H), 4.55–4.50 (m, 1H), 3.89 (dd, J = 10.6,
127.9, 127.8, 122.4, 106.0, 89.9, 75.3, 71.5, 69.1, 65.9, 62.5, 3.8 Hz, 1H), 3.84–3.69 (m, 2H), 3.50–3.39 (m, 2H), 3.10 (dd, J =
56.5, 55.5, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 39.0, 38.4, 36.6, 7.7, 3.3 Hz, 1H), 2.90 (dd, J = 13.6, 3.7 Hz, 1H), 1.11 (s, 3H),
33.8, 33.0, 32.6, 32.3, 30.6, 28.3, 27.5, 25.8, 25.7, 23.6, 23.3, 0.93–0.86 (m, 21H), 0.78 (s, 3H), 0.59 (s, 3H), 0.08 (s, 3H), 0.07
23.0, 20.7, 20.7, 20.6, 18.1, 16.8, 16.6, 15.2; HRMS(ESI): calcd (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.4, 171.9, 143.6,
for [M + H]+ C49H72NO10: 834.5156, found 834.5151.
136.3, 128.3, 127.9, 127.8, 122.4, 102.5, 89.7, 75.1, 74.5, 72.8,
28-O-Benzyl-3-O-[2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D- 65.9, 64.1, 58.4, 55.3, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 38.9,
glucopyranosyl] oleanolic ester (6). To a mixture of 5 (2.39 g, 38.3, 36.6, 33.8, 33.0, 32.6, 32.3, 30.6, 28.2, 27.5, 25.8, 25.6,
2.87 mmol) in pyridine (20 mL), acetic anhydride (0.98 mL, 23.6, 23.3, 23.0, 18.2, 18.1, 16.8, 16.6, 15.2, −5.5, −5.5; HRMS
10.33 mmol) was added. After it was stirred for 1.5 h, the reac- (ESI): calcd for [M + Na]+ C51H81NNaO8 Si: 886.5629, found
tion mixture was quenched by addition of MeOH, filtered and 886.5624.
diluted with CH2Cl2. Then the organic layer was washed with
28-O-Benzyl-3-O-[3,4-O-bi-acetyl-6-O-tert-butyldimethylsilyl-
aqueous HCl (1 N), saturated aqueous NaHCO3 and brine, 2-acetamido-2-deoxy-β-D-glucopyranosyl] oleanolic ester (9).
dried over anhydrous Na2SO4, filtered, and concentrated to To a solution of 8 (1.36 g, 1.57 mmol) in pyridine (20 mL),
afford 6 (2.16 g, 86.3%) as a white foam. Mp 252.1–254.3 °C; acetic anhydride (0.36 mL, 3.77 mmol) was added. After it was
1H NMR (600 MHz, CDCl3) δ 7.38–7.28 (m, 5H), 5.58–5.49 (m, stirred for 2.5 h, the reaction mixture was quenched by
1H), 5.32–5.24 (m, 2H), 5.12–4.98 (m, 3H), 4.67 (d, J = 8.3 Hz, addition of MeOH, filtered and diluted with CH2Cl2. Then the
1H), 4.23 (dd, J = 12.1, 5.5 Hz, 1H), 4.14–4.03 (m, 1H), organic layer was washed with aqueous HCl (1 N), saturated
3.89–3.82 (m, 1H), 3.70–3.64 (m, 1H), 3.07 (dd, J = 11.7, 4.5 Hz, aqueous NaHCO3 and brine, dried over anhydrous Na2SO4, fil-
1H), 2.90 (dd, J = 13.7, 4.0 Hz, 1H), 2.06 (s, 3H), 2.02 (s, 3H), tered, and concentrated to afford 9 (1.28 g, 86.1%) as a white
2.02 (s, 3H), 1.92 (s, 3H), 1.10 (s, 3H), 0.91 (s, 3H), 0.90 (s, 3H), foam. Mp 244.9–246.3 °C; 1H NMR (600 MHz, CDCl3)
0.89 (s, 3H), 0.86 (s, 3H), 0.76 (s, 3H), 0.58 (s, 3H); 13C NMR δ 7.37–7.28 (m, 5H), 5.46 (s, 1H), 5.28 (brs, J = 3.4 Hz, 1H), 5.21
(150 MHz, CDCl3) δ 177.3, 171.0, 170.6, 169.9, 169.4, 143.6, (dd, J = 10.7, 9.3 Hz, 1H), 5.06 (m, 2H), 4.92 (t, J = 9.6 Hz, 1H),
136.3, 128.3, 127.9, 127.8, 122.4, 103.0, 90.6, 72.2, 71.4, 68.9, 4.60 (d, J = 8.2 Hz, 1H), 3.91–3.84 (m, 2H), 3.68–3.61 (m, 1H),
65.9, 62.4, 55.4, 55.3, 47.5, 46.7, 45.8, 41.6, 41.3, 39.2, 38.8, 3.53–3.47 (m, 1H), 3.07 (dd, J = 11.8, 4.4 Hz, 1H), 2.90 (dd, J =
38.3, 36.6, 33.8, 33.0, 32.6, 32.3, 30.6, 27.8, 27.5, 25.7, 25.6, 13.8, 4.0 Hz, 1H), 2.02 (s, 3H), 2.01 (s, 3H), 1.91 (s, 3H), 1.11 (s,
23.6, 23.3, 23.3, 23.0, 20.7, 20.7, 20.6, 18.1, 16.8, 16.4; HRMS 3H), 0.93–0.85 (m, 21H), 0.76 (s, 3H), 0.59 (s, 3H), 0.04 (s, 3H),
(ESI): calcd for [M + Na]+ C51H7 NNaO11: 898.5081, found 0.03 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.4, 171.2, 169.7,
898.5076.
169.3, 143.6, 136.3, 128.3, 127.9, 127.8, 122.5, 103.1, 90.2, 74.6,
28-O-Benzyl-3-O-[2-acetamido-2-deoxy-β-D-glucopyranosyl] 72.7, 69.3, 65.9, 62.8, 55.4, 55.2, 47.5, 46.7, 45.8, 41.6, 41.3,
oleanolic ester (7). To a solution of 6 (2.16 g, 2.47 mmol) in 39.2, 38.8, 38.3, 36.6, 33.8, 33.0, 32.3, 30.6, 29.9, 27.8, 27.5,
CH2Cl2–MeOH (1 : 1, 40 mL), freshly prepared NaOMe in 25.7, 25.6, 25.5, 23.6, 23.3, 23.0, 20.7, 18.2, 18.1, 16.8, 16.4,
MeOH solution (1.0 mol L−1, 0.25 mL) was added. After it was 15.2, −5.5, −5.6; HRMS(ESI): calcd for [M + Na]+ C55H85N-
stirred overnight, the mixture was neutralized with Dowex H+ NaO10Si: 970.5840, found 970.5835.
resin to pH 7, and then filtered. The filtrate was concentrated
28-O-Benzyl-3-O-[2-acetamido-3,4-O-bi-acetyl-6-O-2-deoxy-β-D-
and purified by a silica gel column chromatography (15 : 1, glucopyranosyl] oleanolic ester (10). To a solution of 9 (1.28 g,
CH2Cl2–MeOH) to afford 7 (1.54 g, 83.2%) as a white solid. Mp 1.35 mmol) in THF (25 mL), TBAF (0.39 g, 1.49 mmol) was
146.1–147.3 °C; 1H NMR (600 MHz, CDCl3) δ 7.37–7.27 (m, added. After it was stirred for 11 h, the reaction mixture was
5H), 5.29 (s, 1H), 5.06 (s, 2H), 4.46 (s, 1H), 3.90–3.53 (m, 5H), concentrated and purified by a silica gel column chromato-
3.35 (br s, 1H), 3.05 (s, 1H), 2.89 (t, J = 7.3 Hz, 1H), 1.98 (s, graphy (1 : 1, petroleum ether–EtOAc) to afford 10 (0.80 g,
3H), 1.12 (s, 3H), 0.94–0.86 (m, 12H), 0.75 (s, 3H), 0.59 (s, 3H); 71.2%) as a white solid. Mp 256.7–258.4 °C; 1H NMR
13C NMR (150 MHz, CDCl3) δ 177.5, 172.2, 143.8, 136.6, 128.6, (600 MHz, CDCl3) δ 7.37–7.28 (m, 5H), 5.60–5.50 (m, 1H),
128.1, 128.1, 122.6, 90.2, 75.3, 74.4, 72.2, 70.2, 66.0, 61.9, 55.6, 5.33–5.26 (m, 2H), 5.06 (m, 2H), 4.98 (t, J = 9.6 Hz, 1H), 4.70
47.8, 46.9, 46.0, 41.8, 41.6, 39.5, 39.0, 38.5, 36.8, 34.0, 33.3, (d, J = 8.3 Hz, 1H), 3.86 (dt, J = 10.7, 8.6 Hz, 1H), 3.72–3.67 (m,
32.8, 32.5, 30.8, 28.0, 28.0, 27.8, 26.2, 26.0, 25.8, 23.8, 23.6, 1H), 3.63–3.56 (m, 1H), 3.54–3.49 (m, 1H), 3.09 (dd, J = 11.3,
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5.0 Hz, 1H), 2.90 (dd, J = 13.7, 4.0 Hz, 1H), 2.04 (s, 3H), 2.03 (s, 9H), 0.80 (s, 3H); HRMS(ESI): calcd for [M
3H), 1.92 (s, 3H), 1.10 (s, 3H), 0.94–0.84 (m, 12H), 0.76 (s, 3H), C43H69NNaO12: 814.4717, found 814.4712.
0.58 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.4, 171.0, 170.0,
+
Na]+
Cell viability assay
169.9, 143.6, 136.3, 128.3, 127.9, 127.8, 122.4, 103.0, 90.3, 73.8,
72.2, 69.0, 65.9, 61.6, 55.4, 55.3, 47.5, 46.7, 45.8, 41.6, 41.3, Cell viability was evaluated using an MTT assay. A375 cells
39.2, 38.8, 38.3, 36.6, 33.8, 33.0, 32.6, 32.3, 30.6, 27.8, 27.5, were seeded into 96-well plates at a density of 5 × 104 cells per
25.9, 25.8, 23.6, 23.3, 23.0, 20.7, 20.6, 18.1, 16.8, 16.4, 15.2; well and stabilized at 37 °C for 24 h. Compounds 1 and
HRMS(ESI): calcd for [M + Na]+ C49H71NNaO10: 856.4976, 12a–12f were added to each well at various concentrations (80,
found 856.4970.
40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 and 0.16 μM), and then the
28-O-Benzyl-3-O-[2,3,4-tri-O-benzoyl-α-L-arabinopyranosyl- cells were incubated for 72 h. The MTT solution (20 μL 5 mg
(1→6)-2-acetamido-3,4-O-bi-acetyl-2-deoxy-β-D-glucopyranosy] mL−1) was added to each well, and the cells were incubated for
oleanolic ester (11). A mixture of 10 (130.00 mg, 0.16 mmol) another 4 h. Formazan crystals were dissolved in 150 μL of
and benzoylated arabinose trichloroacetimidate 14 (188.7 mg, DMSO. Cell viability was assessed by measuring the absor-
0.31 mmol) in dry CH2Cl2 (10 mL) was stirred at rt for 30 min. bance at 540 nm wavelength using a microplate reader (BioTek
Then the mixture was cooled to 0 °C and TMSOTf (6.03 μL, ELx800, USA).
0.031 mmol) was added. After stirring at this temperature for
2 h, Et3N was added. The resulting mixture was filtered. The
Cell cycle analysis
filtrates were concentrated to give a residue, which was puri- Cell cycle was assessed using the PI staining assay. A375 cells
fied by a silica gel column chromatography (6 : 1, petroleum were treated with 1 at various concentrations (0, 0.31, 1.25 and
ether–EtOAc) to afford 11 (126.7 mg, 63.6%) as a white foam. 5.0 μM) for 72 h. Then, the cells were collected, washed with
Mp 346.7–348.3 °C; 1H NMR (600 MHz, CDCl3) δ 8.04–7.95 (m, ice-cold PBS buffer, fixed with 80% alcohol at 4 °C for 12 h,
6H), 7.59–7.28 (m, 15H), 5.68–5.64 (m, 1H), 5.60 (d, J = 2.8 Hz, and stained with propidium iodide in the presence of 1%
1H), 5.44 (d, J = 8.9 Hz, 1H), 5.31–5.26 (m, 2H), 5.18 (dd, J = RNAase A at RT (25 °C) for 15 min before analysis by flow
10.7, 9.4 Hz, 1H), 5.12–5.01 (m, 2H), 4.94–4.86 (m, 2H), 4.54 cytometry (BD FACSCalibur USA).
(d, J = 8.3 Hz, 1H), 4.33–4.28 (m, 1H), 3.92–3.82 (m, 3H),
3.77–3.64 (m, 2H), 2.96 (dd, J = 11.7, 4.4 Hz, 1H), 2.90 (dd, J =
Cell apoptosis analysis
13.7, 3.8 Hz, 1H), 2.02 (s, 3H), 2.00 (s, 3H), 1.90 (s, 3H), 1.07 (s, Cells apoptosis was assessed using Annexin V/PI staining
3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H), 0.76 (s, 3H), 0.72 (s, assay. A375 cells were treated with 1 at various concentrations
3H), 0.57 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 177.4, 171.0, (0, 0.31, 1.25 and 5.0 μM) for 72 h. Then, cells were collected,
169.7, 169.4, 165.5, 165.3, 165.1, 143.2, 136.4, 133.3, 130.8, washed with Annexin-binding buffer, and stained with
129.8, 129.8, 129.7, 129.3, 129.1, 128.8, 128.5, 128.4, 128.4, Annexin V fluorescein isothiocyanate (FITC) and PI for 15 min
128.3, 127.9, 127.8, 122.8, 103.2, 99.9, 90.3, 73.5, 72.2, 69.8, at RT (25 °C). After that, the samples were analyzed by flow
69.1, 67.9, 67.4, 65.9, 65.5, 55.4, 55.1, 53.4, 47.4, 46.6, 45.8, cytometry (BD FACSCalibur, USA).
41.5, 41.3, 39.2, 38.9, 38.1, 36.6, 33.8, 33.1, 32.6, 32.3, 30.6,
30.5, 29.6, 27.7, 27.5, 25.8, 25.7, 23.8, 23.6, 23.3, 23.0, 22.6,
Mitochondrial membrane potential assay
20.6, 20.6, 19.1, 18.1, 16.8, 16.3, 15.1, 14.1, 13.6, 7.9; HRMS The mitochondrial membrane potential was assessed using
(ESI): calcd for [M + Na]+ C75H91NNaO17: 1300.6185, found the JC-1 dye. A375 cells were plated at 1 × 106 cells per well in
1300.6191.
24-well plates and incubated with 1 at various concentrations
3-O-[α-L-Arabinopyranosyl-(1→6)-2-acetamido-2-deoxy-β-D- (0, 0.31, 1.25 and 5.0 μM) for 72 h. The cells were subsequently
glucopyranosyl] oleanolic ester (1). A mixture of 11 (126.7 mg, incubated with the JC-1 dye, and finally analyzed by flow
0.099 mmol) and 10% Pd/C (25.3 mg) in EtOAc (5 mL) was cytometry (BD FACSCalibur, USA).
stirred at 50 °C under H2 at atmospheric pressure. After 6 h,
Western blotting
the reaction mixture was filtered and concentrated in vacuo.
Then the residue was dissolved in CH2Cl2–MeOH (1 : 1, 5 mL). A375 cells were seeded at a density of 4 × 105 cells per well and
The resulting solution was added freshly prepared NaOMe in treated with various concentrations of 1 (0, 0.31, 1.25 and
MeOH solution (1.0 mol L−1, 0.1 mL). After it was stirred over- 5.0 μM) for 72 h. After this, cells were collected and washed
night, the mixture was neutralized with Dowex H+ resin to pH twice with ice-cold DPBS. The pellets were resuspended in a
7, then filtered. The filtrate was concentrated and purified by a total protein extraction buffer (20 mM HEPES, 350 mM NaCl,
silica gel column chromatography (7 : 2 : 1, CH2Cl2–MeOH– 20% glycerol, 1% NP-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM
H2O) to afford 1 (59.2 mg, 75.5% in two steps) as a white solid. EGTA, 0.1 mM DTT, 0.1 mM PMSF) containing a protease
1H NMR (600 MHz, Pyr) δ 8.92 (d, J = 9.0 Hz, 1H), 5.45 (d, J = inhibitor cocktail and incubated on ice for 30 min with inter-
3.9 Hz, 1H), 5.00 (d, J = 8.7 Hz, 1H), 4.95 (d, J = 6.6 Hz, 1H), mittent mixing. The protein concentration was measured
4.63–4.51 (m, 2H), 4.48 (dd, J = 8.3, 6.7 Hz, 1H), 4.40–4.30 (m, using the Bradford reagent (Bio-Rad Laboratories Inc, CA,
3H), 4.28–4.22 (m, 1H), 4.21–4.17 (m, 1H), 4.13–4.07 (m, 2H), USA). An equal amount (20 μg) of protein was loaded on 10%
3.77 (d, J = 10.4 Hz, 1H), 3.32 (d, J = 13.7 Hz, 1H), 3.19 (dd, J = polyacrylamide gels and transferred to a nitrocellulose mem-
11.7, 4.4 Hz, 1H), 1.29 (s, 3H), 1.15 (s, 3H), 1.04 (s, 3H), 1.00 (s, brane. After blocking with 5% skimmed milk, the membrane
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was incubated at 4 °C overnight with specific primary anti-
bodies. The membrane was washed and incubated at room
temperature for 1 h with secondary antibodies conjugated with
horseradish peroxidase (HRP). Finally, the immunoblot was
developed for visualization using a chemiluminescence kit.
Primary antibodies for cytochrome c, caspase-9, caspase-3,
caspase-8, Bax, Bcl-2, and β-actin and all secondary antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA,
USA).
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Total RNA was extracted from A375 cells with various concen-
trations (0, 0.31, 1.25 and 5.0 μM) using the Trizol reagent
(Invitrogen). 2 μg of total RNA was used for reverse transcrip-
tion using RevertAid H Minus first strand cDNA synthesis kit
(Fermentas, MA, USA) following the manufacturer’ s instruc-
tions. For real-time qPCR, the ABI PRISM 7900 sequence detec-
tion system (ABI) was used. Nine microliters of master mix
(2 × Maxima SYBR Green/ROXqPCR master mix, 0.3 μM
forward pri mer, 0.3 μM reverse primer) and 1 μL of 100 ng
cDNA were added to the 96-well plates and amplified using a
suitable program. At the completion of cycling, melting curve
analysis was performed to establish the specificity of the
amplicon production. Data were analyzed according to the
comparative Ct method and were normalized by GAPDH
expression. The primers used for qRT-PCR were as follows.
Homo-GAPDH primer (115 bp):
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Forward 5-CATCTTCTTTTGCGTCGCCA-3,
Reverse 5-TTAAAAGCAGCCCTGGTGACC-3;
Homo-Bcl2 primer (81 bp):
Forward 5-ATCCAGGATAACGGAGGC-3,
Reverse 5- CAGCCAGGAGAAATCAAAC-3;
Homo-Bax primer (117 bp):
Forward 5-GACCCGGTGCCTCAGGATGC-3,
Reverse 5-GTCTGTGTCCACGGCGGCAA-3;
Homo-caspase3 primer (139 bp):
Forward 5-CATGGAAGCGAATCAATGGACT-3 ,
Reverse 5-CTGTACCAGACCGAGATGTCA-3.
19 H. C. Wang, J.-H. Yang, S.-C. Hsieh and L.-Y. Sheen,
J. Agric. Food Chem., 2010, 58, 7096–7103.
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Acknowledgements
The authors wish to express their thanks for the financial
support received from the National Natural Science Foun-
dation of China (no. 81273358, 21202103), Program for Innova-
tive Research Team of the Ministry of Education, and Program
for Liaoning Innovative Research Team in University.
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