Synthesis and cytotoxic effect of pseudodiosgenyl saponins with thio-ring F

Article abstract of DOI:10.1016/j.bmcl.2014.01.055

Both the sugar moieties and aglycons of steroid saponins play important roles for their bioactivities. In order to test the biological contribution of the glycosyl residue and search new saponins with notable anticancer activity, mono- and di-saccharide p

Full text of DOI:10.1016/j.bmcl.2014.01.055

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1600–1604
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxic effect of pseudodiosgenyl saponins
with thio-ring F
Xin Zan a,1, Jian Gao a,1, Guofeng Gu b, Shanshan Liu c, Bin Sun a, Lei Liu a, Hong-Xiang Lou a,
⇑
a Department of Natural Product Chemistry, Key Lab of Chemical Biology of MOE (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Wenhua Road, Jinan 250012, PR China
b National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China
c School of Oceanology, Shandong University, Weihai 264209, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Both the sugar moieties and aglycons of steroid saponins play important roles for their bioactivities. In
order to test the biological contribution of the glycosyl residue and search new saponins with notable
anticancer activity, mono- and di-saccharide pseudodiosgenyl saponins 22–28 together with two pseud-
odiosgenyl conjugates 29 and 30 were conveniently synthesized, all of which were based on the aglycon
7 bearing the thio-ring F. The cytotoxicity on human cancer cells (MCF-7, HepG-2, A549) for all of the
synthesized compounds 7 and 22–30 was evaluated by MTT method. The thio-aglycon 7 when
Received 7 October 2013
Revised 3 January 2014
Accepted 18 January 2014
Available online 28 January 2014
Keywords:
conjugated with sugars exhibited potent cytotoxicity, and the introduction of D-glucosamine into aglycon
Steroid saponins
Pseudodiosgenyl conjugates
Cytotoxicity
7 led to the most potent compound 28. Furthermore, DAPI staining, AV/PI staining, AO-relocation,
AO-uptake and LysoTracker Red-uptake assays demonstrated that the cell death caused by neosaponin
28 was at least partially through apoptosis involving lysosomal membrane permeabilization.
Ó 2014 Elsevier Ltd. All rights reserved.
Apoptosis
Steroid saponins, widespread in Chinese medicinal herbs,
possess a wide range of structural diversities and biological activ-
ities, such as cAMP phosphodiesterase inhibition, antifungal, anti-
viral as well as antitumor activities.1–3 Both the sugar moieties
and aglycons play important roles for their bioactivities.4,5 For
example, solamargine, solasonine and khasianine, which have the
identical aglycon but variant sugar moieties showed different
antiproliferative effects.6 Conversely, indioside E and some of its
trisaccharide analogs also exhibited different anticancer activities.7
Solamargine (SM), the most powerful integrant in clinically
antitumor drug BEC (a mixture of glycoalkaloids from the Devil’s
Apple plants),8 comprises a very labile nitrogen ring-F contained
in aglycon, namely salasodine. Consequently, there was surpris-
ingly few reports about the synthesis of SM and its analogs.9 In
addition, the bioisosteres of SM, for example, dioscin and
26-thiodioscin (Fig. 1), also exhibited good anticancer activity.9–
Figure 1. Structures of solamargine, dioscin and 26-thiodioscin.
saponins with pronounced cytotoxic activity and to value the
biological contribution of sugar moieties for saponins. The simpli-
fied residues installed to the aglycon 7 may facilitate industrial-
scale preparation of the biological saponins. Furthermore, the
in vitro anticancer activity of all the synthesized compounds 7 and
22–30 was assayed and the preliminary apoptosis mechanism of
the most potent saponin 28 was also revealed in the manuscript.
As outlined in Scheme 1, synthesis of aglycon 7 was conducted
according to the reported methods.13–16 Ring F opening of dios-
genin 1 with Ac2O/AcCl/Pyr. in refluxing p-xylene, followed by
hydrolysis of the acetyl group gave diol 2 in 79% yield over two
steps.15 The yield of this ring opening reaction was safeguarded
by using a small quantity of Pyr. as a scavenger for the excess acetic
and hydrochloric acid generated from the reaction. Thereafter,
12
So far as we know, there is no saponin except 26-thiodioscin
containing the aglycon 7 with the ring F in which the oxygen
was substituted with sulfur has been reported.12 In view of these
precedents, we herein exploited the synthesis of the pseudodiosge-
nyl conjugates 22–30 containing the aglycon 7 to look for new
⇑
Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382019.
E-mail address: louhongxiang@sdu.edu.cn (H.-X. Lou).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.bmcl.2014.01.055
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

X. Zan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1600–1604
1601
R2
hydrochloride was readily converted to imidate 14 according to
reported method.17 14 was then coupled with aglycon 7 catalyzed
by TMSOTf to give 21. Thereafter, deacetylation with CH3ONa and
hydrolysis of the phthalic anhydride with CH3NH2 provided the
compound 28 in 79% yield over three steps. All of the above
O
O
O
H
H
f
a, b
H
H
H
H
R1O
HO
1
= H, R2 = OH
2
3
4
5
R
1
)
Diosgenin (
c
1
R
= Ts, R2 = OTs
= H, R2 = OTs
= H, R2 = SAc
d
S
S
1
R
e
1
R
O
H
O
H
a
+
R
Br
H
H
H
H
S
S
RO
8
9
HO
31
R = P
R = P
7
8
33
32
R = P
O
g, h
O
H
H
H
34 R = P9
b
9
29
30
H
H
R = S
R = S
H
10
HO
HO
2
O
6
O
7
P8
P9
=
S9
S10
=
AcO
AcO
HO
O
=
=
HO
O
Scheme 1. Reagents and conditions: (a) Ac2O, AcCl, Pyr., p-xylene, reflux 10 h; (b)
KOH, CH3OH/H2O, 79% over two steps; (c) TsCl, Pyr., 0 °C, 12 h, 33% for 4, 55% for 3;
(d) H2O/acetone 3:7, reflux 1.5 h, 84%; (e) CH3COSK, DMF, rt, 24 h, 85%; (f) KOH,
H2O/CH3OH, rt, 12 h, 92%; (g) Zn, AcOH, reflux; (h) KOH, acetone, 90% over two
steps.
OAc
OH
Scheme 3. Reagents and conditions: (a) DIEA, CH2Cl2, rt, 12 h, 85% for 33, 80% for
34; (b) 0.5 N NaOMe, CH2Cl2/CH3OH 2:1, rt, 3 h 90% for 29, and 80% for 30.
Table 1
treatment of 2 with 2.5 equiv p-tosyl chloride (p-TsCl) at 0 °C affor-
The cytotoxicity of compounds 7, 22–30 on three tumor cell lines and one normal cell
ded the desired 26-O-p-tosylpseudodiosgenin
4
(33% yield)
linea
together with 3b,26-di-O-p-toluenesulfonate derivate
3
(55%
Compound
IC50
Cancer cell
(l
M)b
yield), and the undesired 3 was selectively hydrolyzed in refluxing
aqueous acetone for 2 h to give 4 with the 84% overall yield from
2.16 Replacing the 26-O-p-tosyl group in 4 for thioacetyl using
potassium thioacetate in DMF (?5), followed by alkaline hydroly-
sis yielded the disulphide dimer 6.13 Finally, the thio-ring F pseud-
odiosgenin 7 was achieved from 6 through the reaction with zinc
powder in acetic acid and subsequent hydrolysis in 44% overall
yield over eight steps starting from diosgenin 1.
With the key thio-aglycon 7 in hand, we commenced to prepare
the new saponins 22–28 (Scheme 2). In the presence of trimethyl-
silyl trifluoromethanesulfonate (TMSOTf), 7 was glycosylated with
mono- and di-saccharide trichloroacetimidates 8–13 at 0 °C to
afford the protected intermediates 15–20, which was in situ trans-
formed to target neosaponins 22–27 through saponification with
Normal cell
HL7702
MCF-7
HepG-2
A549
7
34.90
14.46
15.23
26.10
20.92
15.47
31.33
6.08
37.67
15.26
11.40
24.42
38.21
15.24
36.07
11.43
>70
30.48
13.57
12.66
21.24
31.37
10.25
32.25
6.03
47.22
36.53
34.63
23.52
51.55
47.30
67.26
31.57
>70
22
23
24
25
26
27
28
29
30
SM
>70
16.37
8.12
>70
10.75
8.18
16.82
2.67
20.71
13.23
a
Values are means of three independent experiments.
IC50 values were determined at 24 h.
b
CH3ONa. En route to compound 28, the commercial
D
-glucosamine
S
S
O
H
O
H
O
CCl3
NH
R
a
H
H
+
H
H
RO
b
HO
1
6
6
1
7
15-20
22-27
R = P -P
R = S -S
8-14
R = P -P
7
1
21 R = P7
b, c
OH
28 R = S7
OH
HO
O
HO
O
O
S1
S5
=
S2
S6
=
=
O
HO
S3
OH
=
S4
OH
=
HO
HO
HO
OH
HO
OH
OH
OH
OH
OH
HO
HO
HO
HO
=
HO
S7
=
O
O
HO
HO
O
O
O
OH
NH2
OH
OH
OBz
O
OBz
O
O
BzO
P1
P5
=
=
P2
P6
=
=
O
P3
OBz
=
P4
=
BzO
BzO
BzO
BzO
BzO
BzO
OBz
OBz
OBz
OBz
OBz
OAc
O
BzO
BzO
OBz
BzO
BzO
O
O
O
P7
=
BzO
AcO
AcO
O
OBz
OBz
NPhth
OBz
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, MS 4 Å, 30 min, 84–88% for 15–21; (b) 0.5 N NaOMe, CH2Cl2/CH3OH 2:1, 3 h, 90–93% for 22–27; (c) aqueous
40% CH3NH2, CH3OH, 4 h, 79% from 21.

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X. Zan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1600–1604
Figure 2. Induction of apoptosis in A549 cells by saponin 28. (A) DAPI staining of A549 cells after 24 h treatment with serial concentrations of 28. (B) Annexin-V/PI uptake of
A549 cells after 28 treatment. (C, D) Quantitation of 28-induced apoptosis in A549 cells by flow cytometric analysis. The values are means standard deviations (indicated by
the bars) of three independent experiments. FL1-H stands for green fluorescence signal received by the photomultiplier tube (PMT), means the amount of apoptotic cells
stained with Annexin V; FL3-H stands for red fluorescence signal received by the photomultiplier tube, means the amount of necrotic cells stained by PI.
resultant glycosidic linkages were stereo-controlled owing to the
neighboring group participation, which were confirmed by 1H
Cytotoxic activity of the synthesized aglycon 7 and novel thio-
pseudodiosgenyl conjugates 22–30 against three cancer cell lines,
including human breast adenocarcinoma cell (MCF-7), human
hepatocellular liver carcinoma cell (HepG-2), and human lung
adenocarcinoma cell (A549), together with the normal cell line hu-
man hepatocyte (HL7702) was assayed by MTT method.21 As
NMR spectra18
(a
glycosidic linkage for 22, J1,2 = 1.2 Hz; 25,
J1,2 = 6.6 Hz. b glycosidic linkages for 23, 24 and 26–28, J1,2 ranging
from 6.2 to 7.1 Hz). The anomeric C-H coupling constants of 22–28
proved conformation of their glycosyl residues as follows: 22, 1C4,
1
1JC,H = 171 Hz; 23–28, 4C1, JC,H ranging from 161 to 163 Hz.
shown in Table 1, Saponin 28 bearing
residue displayed the strongest antiproliferative activity against
MCF-7 and A549 cell lines with IC50 of 6.03 and 6.08 M,
respectively, whereas 28 was less cytotoxic to the normal
HL7702 (IC50 = 31.57 M). Compounds 22, 23 and 26, containing
-rhamnose, -xylose, and -galactose, respectively, exhibited
moderate cytotoxicity on three cancer cell lines with IC50 ranging
from 10.24 to 15.47 M and weak antiproliferative effect to
D-glucosamine as sugar
With hope to simplify the structure of sugar residue and to
expurgate the unnecessary glycosidic linkage, two pseudodiosge-
nyl conjugates containing a chain with free hydroxyl groups were
designed and assembled (Scheme 3). Coupling of 7 with 31 and 32,
prepared from 1,3-dioxolane and 1,3-dioxolan-4-yl-methanol,19,20
followed by deprotection provided the compounds 29 and 30,
respectively.
l
l
L
D
D
l

X. Zan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1600–1604
1603
Figure 3. The effect of saponin 28 on lysosomal membrane permeabilization was evaluated with AO relocation analysis, AO-uptake and LysoTracker Red uptake methods. (A)
Lysosomal rupture measured by AO relocation analysis. Increase in green-AO fluorescence, indicating AO was released from ruptured lysosomes to the cytosol, and was
detected by flow cytometry. The values are means standard deviations (indicated by the bars) of three independent experiments. (B) Lysosomal rupture was measured by
AO-uptake and LysoTracker Red uptake and visualized by fluorescence microscopy. FL1-H stands for Green Fluorescence signal received by the photomultiplier tube (PMT),
means the amount of AO is present in the cytosol.
HL7702 (>30
l
M). In addition, compound 30 containing a chain
apoptosis. Necrotic cells can be stained by PI, and exhibited red
fluorescence. As shown in Figure 2C and D, the proportion of apop-
totic cells stained with Annexin V increased after the treatment of
28. All of the above results suggested that the cytotoxicity of
compound 28 against A549 cells was at least partly induced by
the apoptosis. It is noteworthy that the 28-induced anticancer
activity on A549 could be only partially the result of apoptosis,
and the oncosis may be also involved, which is actually our
ongoing research project.
with two free hydroxyl groups also exhibited the comparable anti-
cancer effect with compounds 22, 23 and 26. The other saponins
24, 25, and 27 showed a little better anticancer effect than aglycon
7. Compound 29 did not show the antiproliferative effect against
the three cancer cells at a concentration of 70
indicated that introducing glycosyl residue to aglycon 7 could im-
prove its cytotoxicity, and incorporating -glucosamine to 7 leaded
to the most potent neosaponin 28. Incorporation of glycosyl
residue to the aglycon 7 might be advantageous for the uptake of
compounds into cell. Furthermore, the enhanced alkalinity of
lM. These results
D
To assess the effect of 28 on lysosomal membrane permeabili-
zation, three assays including AO-relocation, AO-uptake and
LysoTracker Red-uptake were applied.25,28 AO, a metachromatic
fluorophore that accumulates in normal lysosomes, exhibits a
low level of green fluorescence when cloistered inside of the
lysosome. When lysosomes are disrupted, AO is present in the
cytosol, manifesting a much brighter green fluorescence.29 AO relo-
cation method demonstrated that lysosomes had been disrupted
since enhanced cytosolic green fluorescence was detected by flow
cytometry after AO-loaded cells were exposed to 28 for 2 h
(Fig. 3A). AO-uptake method verified the results of AO relocation
method by showing decreased numbers of AO-accumulating lyso-
somes after 2 h exposure to 28. The loss of the punctate red spots
(lysosomes with intact integrity) depended on the concentrations
of 28 (Fig. 3B). LysoTracker Red-uptake methods also demon-
strated lysosomal destabilization by showing lysosomes became
swollen and trended rupture after 2 h exposure to 28, while the
nucleus stained with DAPI appeared apoptotic characteristics
(Fig. 3B). These results show that compound 28 caused lysosomal
vacuolation, disrupted lysosomal membrane integrity and initiated
an early lysosomal destabilization pathway leading to apoptosis.
In summary, nine pseudodiosgenyl conjugates containing the
aglycon 7 with a thio-ring F were facilely assembled. Preliminary
anticancer evaluation indicated that the incorporation of glycosyl
glycoside 28 after the introduction of D-glucosamine might facili-
tate its accumulation in protonated form within acidic vesicles
(lysosomes), which could induce lethal lysosomal destabilization
and cell death.22
As we know, two types of prelethal reaction of cell death have
been characterized: apoptosis and oncosis.23,24 In addition, our
group has demonstrated that some biological saponins, for exam-
ple, SM and indioside E, could cause cell death through both of
these two patterns.7,10,25 Encouraged by the good anticancer
activity and lower toxicity to normal cell HL7702 of neosaponin
28, we performed further test to elucidate whether its cytotoxic
effect of A549 was associated with apoptosis or not. After A549
cells were incubated with 2, 4 and 6 lM concentrations of neosa-
ponin 28 for 24 h, apoptotic characteristics were observed by DAPI
staining.26 Morphological changes, such as chromatin condensa-
tion, cell shrinkage, membrane blebbing, and apoptotic body
formation, were canonical apoptotic markers (Fig. 2A and B). In
addition, A549 cells double stained with Annexin V and propidium
iodide (PI) were analyzed by flow cytometry to quantitate the
extent of 28-induced apoptosis.27 Annexin V staining provides
the possibility to detect the presence of phosphatidylserine on
the outer leaflet of the cell membrane, a characteristic related to

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X. Zan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1600–1604
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D
the most potent neosaponin 28. Compound 28-induced apoptosis
on A549 cell was involved lysosomal vacuolation and disruption
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This work was supported by grants from Natural Science Foun-
dation of China (No. 81172956).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.
055.
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