Efficient synthesis of trisaccharide saponins and their tumor cell killing effects through oncotic necrosis

Article abstract of DOI:10.1016/j.bmcl.2010.12.046

To investigate the relationship of cytotoxicity and saponins with varied aglycones, based on the structure of indioside E 1, a natural derived anti-tumor active ingredient from Chinese medicinal plant Solanum indicum L., five novel saponins 2-6 bearing th

Full text of DOI:10.1016/j.bmcl.2010.12.046

Bioorganic & Medicinal Chemistry Letters 21 (2011) 622–627
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of trisaccharide saponins and their tumor cell killing
effects through oncotic necrosis
Jian Gao a, Xia Li b, Guofeng Gu c, Bin Sun c, Min Cui b, Mei Ji a, Hong-Xiang Lou a,
⇑
a Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan 250012, PR China
b School of Oceanology, Shandong University, Weihai 264209, PR China
c National Glycoengineering Research Center, Shandong University, 44 West Wenhua Road, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To investigate the relationship of cytotoxicity and saponins with varied aglycones, based on the structure
of indioside E 1, a natural derived anti-tumor active ingredient from Chinese medicinal plant Solanum
indicum L., five novel saponins 2–6 bearing the same trisaccharide moiety together with 1 were efficiently
synthesized via a transglycosylation strategy. MTT assay revealed the killing effects to tumor cells of the
synthesized saponins are varied with the change of aglycones. Furthermore, time-lapse microscopy, LDH
release, PI staining, and immunocytochemical investigations demonstrated that the cell death caused by
neosaponin 2 was through oncotic necrosis involving plasma membrane perturbation and destruction of
cytoskeleton.
Received 16 November 2010
Revised 7 December 2010
Accepted 8 December 2010
Available online 13 December 2010
Keywords:
Saponin
Indioside E
Trisaccharide analogues
Cytotoxicity
Oncosis
Ó 2010 Elsevier Ltd. All rights reserved.
Natural derived saponins from terrestrial plants or marine organ-
isms display a wide spectrum of structural diversity and versatile
biological activities, which include anti-inflammatory, antibacterial,
antiparasitic, antifungal, and anti-tumor activities.1–5 Their biologi-
cal effects were correlated to both their sugar residues and agly-
cones.6,7 To exemplify the structure-activity relationship study,
solamargine and solasonine, which have same aglycone but differ-
ent composition of sugar moiety, showed different tumor cell killing
results while OSW-1 and some of its disaccharide analogues, which
have same sugar moiety but variant aglycones showed comparable
anticancer activity.8–11
with pronounced anticancer activity. Furthermore, the cytotoxicity
of all the synthesized saponins 1–6 was assayed and the oncotic
necrosis mechanism of the potent active compound was also
revealed in present study.
Compound
1 has been previously synthesized using six
straightforward sequential reactions,13 the target neosaponins
were assembled here by an efficient transglycosylation strategy,
using the trisaccharide donor directly transferred to different agly-
cones. As shown in Scheme 1, isopropyl 3-O-fluorenylmethoxycar-
bonyl-4,6-di-O-benzylidene-1-thio-b-
regioselectively prepared from commercially available isopropyl
b- -thiogalactopyranoside (IPTG) according to the published
procedure in two steps.14 Thereafter, standard glycosylation of
galactopyranosyl acceptor 7 with 2,3,4-tri-O-acetyl- -rhamno-
pyranosyl trichloroacetimidate in anhydrous CH2Cl2 using
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as catalyst
provided -(1?2)-linked disaccharide 9, followed by triethyl-
D-galactopyranoside 7 was
Indioside E (1, Fig. 1), namely diosgenyl
a
-L
-rhamnopyranosyl-
D
(1?2)-[b- -xylopyranosyl-(1?3)]-b- -galactopyranoside, was ex-
D
D
tracted from a traditional Chinese medicinal plant Solanum indicum
L. herb.12 Preliminary biological test showed that 1 exhibits prom-
ising cytotoxic activity toward human hepatocellular carcinoma
BEL-7402.13 Attracted by its potent anti-tumor activity and the
importance of sugar residues for saponins, we exploited here the
practical and efficient synthesis of indoiside E and its novel
analogues 2–6 by introducing different aglycones with the same
trisaccharide into the structure (Fig. 1) for searching new saponins
a-L
8
a
amine (TEA) promoted removal of Fmoc group on 3-OH of galact-
ose unit of 9, furnished the disaccharide acceptor 10 in excellent
yield of 84% for two steps. Subsequently, under the promotion of
TMSOTf, coupling of disaccharide acceptor 10 with benzoyl-pro-
tected xylosyl imidate 11 afforded thiotrisaccharide 12 as a potent
glycosylation donor in 67% yield. It is noteworthy that the xylosyl
residue in 12 adopts a 1C4 conformation instead of the typical 4C1
form. This conformational assignment was deduced from the small
coupling constants (J1,2 = 3.0 Hz, J2,3 = J3,4 = 3.6 Hz) observed in 1H
NMR spectrum. The chemical shift of C-1 (d 99.7 ppm) in 13C
⇑
Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382019.
E-mail address: louhongxiang@sdu.edu.cn (H.-X. Lou).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.046

J. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 622–627
623
Figure 1. Structures of indioside E (1) and synthesized indioside E trisaccharide derivatives (2–6).
NMR spectrum confirmed the b-bond linkage between xylose and
galactose residue in 12.
clo[5.4.0]undec-7-ene (DBU) in dichloromethane. As expected,
trisaccharide imidate 14 was successfully and efficiently
transferred to the varied aglycones (e.g., diosgenin, tigogenin,
cholesterol, sitosterol, taraxerol, and lupeol) in the presence of
TMSOTf at 0 °C to afford the corresponding protected b-neoglyco-
sides 15–20 in high yields of 81–87% as well as trace amount of
Subsequently, thiotrisaccharide 12 was used as a glycosyl donor
to transfer to aglycone by standard glycosylation. Unfortunately,
direct glycosylation of diosgenin with 12 under the promotion of
N-iodosuccinimide (NIS)-TMSOTf was troublesome. The reaction
resulted in a complex mixture of products and the desired pro-
tected glycosides with poor yield and can only be detected by
MS analysis. A detailed evaluation of the result suggested that thio-
glycoside 12 was not an active glycosyl donor matching 3-OH of
diosgenin alycone in this glycosylation. To circumvent this prob-
lem, compound 6 was converted into more efficient trichloroace-
timidate 14 in 81% yield for two steps as follows: (i) NIS and
HClO4 immobilized on silica (NIS/HClO4-silica) promoted chemo-
selective hydrolysis of thioglycoside 12 in CH2Cl2–H2O co-solvent
system (?13);15,16 (ii) trichloroacetimidate activation of anomeric
carbon using trichloroacetonitrile (CCl3CN) and 1,8-diazabicy-
their
a
isomers.17 Interestingly, the stereo-selectivity of this glyco-
sylation was predominantly b stereo-controlled without the neigh-
boring group participation effect of donor 14, which was adjusted
by the coupling constants of the galactosyl H-1s (J1,2 = ꢀ7.8 Hz) in
1H NMR spectra and the chemical shifts of C-1s (d ꢀ100.2 ppm).
Global deprotection of pure b-protected glycosides 15–20 was
accomplished in aqueous 80% acetic acid at 70 °C, followed by
1 N NaOH to furnish the target indoiside E 1 and noesaponins
2–6.18 The conformation of xylosyl residues in compounds 1–6
were converted back to 4C1 form, supported by the doublet peaks
of xylosyl H-1s at ꢀ5.05 ppm (J1,2 = ꢀ7.8 Hz) in 1H NMR spectra.
Table 1
The cytotoxicity of compounds 1–6 on four tumor cell lines
Compound
IC50 (lM)
K562
KB
MCF-7
U87
1
2
3
4
5
6
3.74
3.77
11.35
>40
>40
>40
1.71
1.32
16.28
>40
>40
>40
1.82
2.61
9.48
>40
>40
>40
2.48
3.12
16.48
>40
>40
>40

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J. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 622–627
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 45 min, 92% for 9, 67% for 12; (b) Et3N, rt, 91%; (c) diosgenin, NIS-TMSOTf, ꢁ40 °C to rt; (d) NIS-HClO4/SiO2,
CH2Cl2/H2O 10:1, 0 °C, 1.5 h; (e) CCl3CN, DBU, CH2Cl2, 0 °C, 2 h, 81% from 12; (f) aglycones, TMSOTf, CH2Cl2, 0 °C, 40 min: diosgenin for 15, 87%; tigogenin for 16, 81%;
cholesterol for 17, 85%; sitosterol for 18, 82%; taraxerol for 19, 82%; lupeol for 20, 87%; (g) aqueous 80% AcOH, 70 °C, 3 h; (h) 1 N CH3ONa, CH3OH/CH2Cl2 2:1, 3 h.
The cell killing effects of above synthesized saponins 1–6 on
K562, KB, MCF-7 and U87 cells and three normal cell lines
(HL7702, H9C2, and EVC304) was assayed using MTT method. As
shown in Tables 1 and 2, both 1 and 2 displayed strong cell killing
effects with IC50 ranging from 1.32 to 3.77 lM, but 2 had a lower
cytotoxicity to normal hepatocyte cell HL7702. Compound 3 exhib-
and taraxerol, lupeol, and sitosterol replacement will decrease
the cytotoxicity.
The excellent anticancer activity and lower toxicity to HL7702 of
neosaponin 2 encouraged us to investigate the possible mechanism
of its action at the molecular level. Inspection of 2-treated cells by
means of time-lapse microscopy revealed the presence of cells swell
and membrane surface blebs. Figure 2 showed that the blebs of K562
and MCF-7 formed as early as 30–60 min post-treatment with 2 and
blebs last 15–30 min in these two cells. However, the cell swell last
longer time and membrane disrupt at the end. The above results
ited a moderate anti-tumor activity despite it is not so toxic to nor-
mal cells. Meanwhile, neosaponins 4–6 were inactive at
a
concentration of 40 M. The results indicated that the spiros-
l
tane-type aglycone part of 1 was of importance for its activity
Table 2
Cytotoxicity of 1–3 on normal cell lines
Compound
IC50 (lM)
EVC304
HL7702
H9C2
1
2
3
3.93
4.03
>20
4.81
6.92
>20
>20
>20
>20

J. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 622–627
625
Figure 2. Time-lapse analysis of cell morphological changes and membrane blebs formation induced by 5 lM 2 in K562 and MCF-7 cells.
indicated compound 2 exhibited morphologic oncotic feature and
resulted the damage of plasma membrane.
Plasma membrane integrity was monitored by measuring the
release of the lactose dehydrogenase (LDH) into the incubation
medium. The data showed that LDH release of K562 and MCF-7
The conformations of 1 and 2 were energy-minimized and super-
imposed, and the results are shown in Figure 5.22 Obviously, there is
a striking overlap of the calculated structures of 1 and 2 except for a
few minor differences in their aglycones owing to the double bond of
1. In consideration of the promising anticancer activity and lower
toxicity to HL7702 of 2, we can infer that the double bond of 1
may be a good active spot to modify in our next work.
cells were increased after 10 min treatment of 5
and peaked at about 180 min (Fig. 3A).
lM compound 2
The integrity of the cell membrane was also determined by the
uptake of the PI (DNA intercalating fluorescent dye).19 Staining of
the cells with PI and subsequently analyzed by fluorescence
microscopy. As shown in Figure 3B, the control cells unstained
by PI and the cells treated with compound 2 stained by PI since
the cell membrane was disrupted.
Several studies have suggested that surface bleb formation is re-
lated to alteration in cytoskeleton network.20,21 Here, immunocy-
tochemical investigations revealed the change of cytoskeleton
network in compound 2-exposed cells. As shown in Figure 4, the
loss of microtubules and actins microfilaments were observed in
2-treated cells. The results demonstrated that 2 induced plasma
membrane blebs, which was related to its destroying microtubules
and actins in culture cells.
In summary, indioside E 1 together with its five neosaponins 2-
6 bearing the same trisaccharide residue were synthesized via an
efficient transglycosylation strategy. A preliminary SAR analysis
indicated that the spirotane-type aglycones of 1 and 2 are of value
for their anticancer activity, and replacing diosgenin with
tigogenin decrease cytotoxicity toward HL7702 without loss of
anti-tumor activity. Furthermore, 2-induced oncosis in K562 and
MCF-7 cells is associated with both plasma membrane permeabil-
ity and the disruption of cytoskeleton network. Collectively, our
study suggest that 2 exhibits potent anti-tumor activity via a novel
form of oncosis-like cell death and may merit further investigation
as a potential therapeutic agent for cancer treatment. Much more
work about the biochemical mechanism by which 2-induced onco-
sis is required and is ongoing.

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J. Gao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 622–627
Figure 3. (A) 2-induced LDH release from K562 and MCF-7cells. (B) PI uptake of K562 and KB cells after 2 treatment.
Figure 4. Effect of 2 on the microtubule and actin distribution in MCF-7 cells analyzed by a fluorescent microscope at magnification of 1000ꢂ.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.046.
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lupeol.) and trichloroacetimidate 14 (100 mg, 0.089 mmol) was dissolved in
anhydrous CH2Cl2 (4 mL). To the solution was added TMSOTf (1.6
lL, 9
lmol)
C
44H72O16Na (M+Na+): 879.4713; found 879.4719.
at 0 °C under N2 protection. The reaction mixture was stirred for 40 min,
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b isomer with the yield ranging from 81% to 87%.
a
5:1 solution of
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18. Physical and analytical data for neosaponin tigogenyl 3-O-{
a-L-rhamnopy-
ranosyl-(1?2)-[b- -xylopyranosyl-(1?3)]-b- -galactopyranoside} (2).
D
D
½ ꢃ
a 2D0