Efficient synthesis and antitumor activities of indioside e analogs

Article abstract of DOI:10.1080/07328303.2014.900564

An efficient synthesis of seven analogs of a naturally occurring saponin, indioside E, is described. The synthesis used a 3,4,6-O-protected D-galactopyranosyl thioglycoside as the key intermediate to enable regioselective glycosylation and one-pot multistep reactions. Antitumor activities of the synthetic saponins against a human hepatocellular carcinoma cell line BEL-7402 and a human breast adenocarcinoma cell line MCF-7 were evaluated by means of the CCK-8 assay. Analogs carrying trisaccharides with the D-xylopyranose in indioside E substituted with a D-ribopyranose and an L-arabinopyranose or with its diosgenin aglycon replaced with tigogenin exhibited similar antitumor activities as indioside E, but not other analogs.

Full text of DOI:10.1080/07328303.2014.900564

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Efficient Synthesis and Antitumor
Activities of Indioside E Analogs
Yisheng Zhaoab, Lian Anbc, Guofeng Guab & Zhongwu Guob
a School of Pharmaceutical Science, Shandong University, Jinan
25012, PR China
b National Glycoengineering Research Center, Shandong University,
Jinan 250010, PR China
c School of Life Science, Shandong University, Jinan 250100, PR
China
Published online: 03 Apr 2014.
To cite this article: Yisheng Zhao, Lian An, Guofeng Gu & Zhongwu Guo (2014) Efficient Synthesis and
Antitumor Activities of Indioside E Analogs, Journal of Carbohydrate Chemistry, 33:3, 152-168, DOI:
10.1080/07328303.2014.900564
To link to this article: http://dx.doi.org/10.1080/07328303.2014.900564
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Journal of Carbohydrate Chemistry, 33:152–168, 2014
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2014.900564
Efficient Synthesis
and Antitumor Activities
of Indioside E Analogs
Yisheng Zhao,1,2 Lian An,2,3 Guofeng Gu,1,2 and Zhongwu Guo2
1School of Pharmaceutical Science, Shandong University, Jinan 25012, PR China
2National Glycoengineering Research Center, Shandong University, Jinan 250010, PR
China
3School of Life Science, Shandong University, Jinan 250100, PR China
An efficient synthesis of seven analogs of a naturally occurring saponin, indioside
E, is described. The synthesis used a 3,4,6-O-protected D-galactopyranosyl thioglyco-
side as the key intermediate to enable regioselective glycosylation and one-pot multi-
step reactions. Antitumor activities of the synthetic saponins against a human hep-
atocellular carcinoma cell line BEL-7402 and a human breast adenocarcinoma cell
line MCF-7 were evaluated by means of the CCK-8 assay. Analogs carrying trisaccha-
rides with the D-xylopyranose in indioside E substituted with a D-ribopyranose and an
L-arabinopyranose or with its diosgenin aglycon replaced with tigogenin exhibited sim-
ilar antitumor activities as indioside E, but not other analogs.
Keywords Saponin; Indioside E; Thioglycoside; One-pot synthesis; Antitumor
INTRODUCTION
Indioside E (1) (Fig. 1), a diosgenyl triglycoside extracted from the Solanum
indicum L. plant that has long been used as a traditional anti-inflammatory
medicine in China,[1] has displayed promising cytotoxic activity toward human
hepatocellular carcinoma BEL-7402.[2] Further investigation on the structure-
activity relationships of indioside E and its derivatives containing various
aglycons indicated that the spirotane type of aglycon was essential for its
anticancer activity.[3] For example, neosaponin 2 (Fig. 1), an analog of 1 that
had the diosgenin glycon replaced with tigogenin, exhibited very good cyto-
static activities against various tumor cells while showing low toxicity to nor-
mal hepatocyte cell HL7702.[3] Time-lapse microscopic, immunocytochemical,
Received January 14, 2014; accepted February 28, 2014.
Address correspondence to Guofeng Gu, School of Pharmaceutical Science, Shandong
University, Jinan 25012, PR China; and National Glycoengineering Research Center,
Shandong University, Jinan 250010, PR China. E-mail: guofenggu@sdu.edu.cn
152

Indioside E Analogs and Their Antitumor Activities
153
Figure 1: The structures of indioside E (1) and neosaponin (2).
and many other studies have demonstrated that the cytostatic activity of 2 was
due to plasma membrane perturbation and cytoskeleton destruction.[3]
In addition to aglycon, the carbohydrate moiety of saponins may also play
an important role in their biological activities. For instance, structure-activity
relationship studies on dioscin (diosgenin 3-O-chacotrioside) and its deriva-
tives revealed that its trisaccharide moiety was essential for the cytotoxic ac-
tivity.[4–6] The cell apoptosis activity of solasodine glycoalkaloids was mainly
due to the rhamnose residue in their sugar chains.[7] Thus, the influence of
the glycan structure in indioside E on its bioactivity is worth investigating. In
this regard, we designed and synthesized herein a series of diosgenyl and ti-
gogenyl saponins 3a–c and 4a–c (Fig. 2), in which the D-xylopyranose residue
on the galactose 3-O-position of the glycan in 1 and 2 was substituted with
D-lyxopyranose, D-ribopyranose, and L-arabinopyranose, while the rhanmose
residue remained unchanged, considering the important impact of rhanmose
on the biological activities of other saponins.[7,8] The antitumor activities of the
synthetic saponins 2–4 against two human cancer cell lines, BEL-7402 and
MCF-7—a human breast adenocarcinoma cell—were evaluated by in vitro an-
tiproliferative assays.
RESULTS AND DISCUSSION
In the past few decades, many practical and efficient synthetic methods have
been developed for saponins.[9–13] Recently, we prepared several saponins that
Figure 2: Designed indioside E derivatives, 3a–c and 4a–c, to be studied in this research.

Y. Zhao et al.
154
contained 2-O- and 2,4-O-branched oligosaccharides by a one-pot multistep
glycosylation strategy, which used a partially protected thiogalactoside as the
key building block.[8,14] This efficient protocol was then explored in the synthe-
sis of target molecules 2–4. As outlined in Scheme 1, disconnecting the gly-
cosidic linkage at the galactose 3-O-postion would afford two common inter-
mediates 5a and 5b containing the same disaccharide moiety, which could be
readily prepared in a one-pot three-step manner upon chemoselective galacto-
sylation of aglycons 7a and 7b with partially protected thiogalactoside 8, 2-O-
rhamnosyaltion of the resultant intermediate with trichloroacetimidate 9, and
then removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) group with triethy-
lamine (TEA). The use of 8 to facilitate a one-pot three-step assembly protocol
would significantly simplify this synthesis by eliminating a deprotection step
and multiple steps of column purification.
Scheme 1: Retrosynthetic plan for target molecules 2–4.
The synthesis of neosaponin 2 is shown in Scheme 2. First, disaccha-
ride glycosides 5a[2] and 5b were prepared by a one-pot multistep protocol
in very good overall yields, 68% and 65%, respectively. This involved direct
glycosylation of diosgenin 7a and tigogenin 7b with 8[15] by the treatment of
N-iodosuccinimide (NIS) and trimethylsilyl triflate (TMSOTf) at –40◦C, fol-
lowed by rhamnosylation of the resulting saponin intermediate with 9 in the
presence of TMSOTf at 0◦C and TEA-promoted removal of the Fmoc group on
the galactose 3-O-position. The coupling constants of the galactosyl H-1 signal

Indioside E Analogs and Their Antitumor Activities
155
(J1,2 = 7.2 Hz) at δ 4.48 ppm and the rhamnosyl H-1 signal (J1,2 = 1.6 Hz)
1
at δ ∼5.32 ppm in the H NMR spectra of 5a and 5b confirmed their correct
glycosidic configurations. No trace of self-condensed products was detected in
our experiments. Moreover, such high glycosylation efficiency in condensation
of steroidal aglycone with a free 2-OH galactosyl thioglycoside could be ratio-
nalized as formation of 1,2-anhydrosugar intermediate presumed by Linhardt
et al.[15] Next, glycosylation of 5b with trichloroacetimidate 6a in the pres-
ence of TMSOTf furnished the fully protected trisaccharyl saponin 10[3] in a
very good yield (71%). Finally, global deprotection of 10 was accomplished in
two steps, including removal of the benzylidene group using 80% HOAc and
saponification of the resulting intermediate with NaOMe in methanol, which
afforded the target molecule 2 in a 93% yield. It is worth mentioning that the
small proton coupling constants (J1,2 < 1.0, J2,3 = J3,4 = 3.2, J4,5a = 1.8 Hz)
of xylosyl residue in 10 revealed that the benzoylated D-xylopyranose residue
1
in 10 adopted a C4 conformation, whereas after complete deprotection the
4
xylopyranose residue in 2 resumed the C1 conformation (H-1Xyl: δ 4.40 ppm,
J1,2 = 6.6 Hz), which was consistent with previously reported data.[2,3]
Scheme 2: Synthesis of neosaponin 2.
Saponins 3a–c and 4a–c were prepared by a similar strategy (Sch. 3). Gly-
cosylation of 5a with imidates 6b–d in the presence of a catalytic amount
of TMSOTf at 0◦C afforded fully protected trisaccharyl saponins 11a–c in
good yields (62%–76%). Likewise, glycosylation 5b with 6b–d gave 12a–c
(61%–78%). Subsequently, 11a–c and 12a–c were globally deprotected in two
steps according to the protocol described above to furnish target molecules
3a–c and 4a–c (92%–96%). The NMR spectra of 11 and 12 suggested that the
benzoylated ribopyranose and arabinopyranose residues in 11b,c and 12b,c
adopted a 1C4 conformation. Interestingly, after global deprotection, while the
arabinose residue in 3c and 4c was converted to the 4C1 conformation (H-1Ara
:
δ 4.40 ppm, J1,2 = 7.0 Hz), the ribose residue in 3b and 4b kept the 1C4 confor-
mation (H-1Rib: δ 4.92 ppm, J1,2 = 3.9 Hz).

Y. Zhao et al.
156
Scheme 3: Synthesis of target molecules 3a–c and 4a–c.
The antiproliferative activities of synthetic saponins 2–4 against two hu-
man tumor cell lines, BEL-7402 and MCF-7, were assessed according to the
standard protocol of the CCK-8 assay[16] with natural indioside E (1) as a
positive control. The results are shown in Table 1. As reported,[2,3] 1 exhib-
ited strong antiproliferative activities against both tumor cell lines with half-
maximal (50%) inhibitory concentrations (IC50) of 3.76 μM for BEL-7402 and
2.93 μM for MCF-7. The antitumor activities of 2, the ribosyl derivative 3b,
Table 1: Antiproliferative activities of 1–4 against tumor cell lines BEL-7402 and
MCF-7
IC50 (μM)a
Compound
BEL-7402
MCF-7
Indioside E (1)
3.76 0.59
4.29 0.61
10.61 1.65
3.37 0.42
4.67 0.63
6.35 0.51
5.42 0.33
7.89 0.76
2.93 0.77
3.20 0.06
9.33 0.94
3.22 0.56
3.99 0.37
7.06 0.55
4.94 0.73
9.00 0.86
2
3a
3b
3c
4a
4b
4c
a IC50 values are the mean results of three independent experiments.

Indioside E Analogs and Their Antitumor Activities
157
and the arabinosyl derivative 3c were comparable to that of 1, while other
synthetic saponins exhibited a two- to three-fold decrease in antiprolifera-
tive activity against both tumor cell lines. These results indicate that the
xylose residue of trisaccharide moiety in 1 plays a critical role in its antitu-
mor activity. In particular, when the D-xylose residue was replaced with D-
lyxose, the antitumor activities of the resulting 3a and 4a were significantly
reduced. It seemed that, although the spirotane type of aglycon in 1 was crit-
ical for its anticancer activity,[3] some small change in the aglycon structure
had little influence on its antitumor activity, because 2 had similar activ-
ity as 1. On the other hand, comparing the activities of 3b to 4b and 3c to
4c, which were only different in their aglycon structures, led us to suggest
that after the oligosaccharide in 1 was changed, the aglycon structure did
have some impact on the biological activities. Evidently, depending on spe-
cific situations, both the glycan and the aglycon may affect the function of a
saponin.
In summary, seven analogs of naturally occurring indioside E bearing dif-
ferent aglycons and glycans were synthesized by a highly efficient strategy
based on one-pot multistep glycosylation. In vitro biological assays of the syn-
thetic analogs against tumor cell lines BEL-7402 and MCF-7 revealed that
the glycan in indioside E had a big impact on its antitumor activity, although
some small change in the aglycon structure (e.g., 1 vs. 2) had little influence
on the antitumor activity. However, after the glycan was changed, the aglycon
structure did matter significantly. Combined with previous observations,[3] the
results have demonstrated that both the carbohydrate moiety and the aglycon
play an important role in the biological functions of saponins, whereas the ac-
tion mechanisms of these saponins and the exact roles that the carbohydrate
and the aglycon play in the function are interesting questions that are worth
further investigations.
EXPERIMENTAL SECTION
General Methods
Optical rotations were determined at 20◦C with a Rudolph Autopol I auto-
1
matic polarimeter. H and 13C NMR spectra were recorded with a Bruker 400
or a Varian 600 MHz spectrometer for solutions in CDCl3 or CD3OD. Chem-
ical shifts (δ) are given in ppm downfield from internal Me4Si. Positive-mode
electrospray ionization (ESI) high-resolution mass spectroscopy (HRMS) was
recorded on a JEOL JMS-DX-303HF spectrometer. Thin-layer chromatography
(TLC) was performed on silica gel HF254 plates with detection by charring with
30% (v/v) H2SO4 in MeOH or by a UV detector. Column chromatography was
conducted by elution of a silica gel column with mixtures of ethyl acetate and

Y. Zhao et al.
158
petroleum ether (b.p. 60–90◦C) as the eluents. Solutions were concentrated at
<60◦C under diminished pressure.
General Procedure for One-Pot Synthesis of Disaccharide
Saponins 5a and 5b
To a solution of steroidal aglycon 7a or 7b (1.0 mmol) and thioglyco-
side 8 (1.2 mmol) in anhydrous CH2Cl2 (10 mL) were added NIS (320 mg,
1.42 mmol) and TMSOTf (27 μL, 0.15 mmol) at –40◦C under an N2 atmo-
sphere. The reaction mixture was stirred under this condition for 30 min,
when TLC (3:1 petroleum ether-EtOAc) indicated the complete consump-
tion of 7. The reaction mixture was warmed to 0◦C, and then a solution of
trichloroacetimidate 9 (1.5 mmol) in anhydrous CH2Cl2 (3 mL) was quickly
injected into the reaction mixture, which was followed by the addition of
TMSOTf (9 μL, 0.05 mmol). The resulting mixture was stirred for another
1 h, at which time TLC (2:1 petroleum ether-EtOAc) indicated the comple-
tion of reaction. The reaction mixture was diluted with CH2Cl2 (50 mL) and
then quenched with triethylamine (3.0 mL, 21 mmol). The resulting mixture
was stirred at rt for 1.5 h and then concentrated to dryness under reduced
pressure. Purification of the residue on a silica gel column with petroleum
ether-EtOAc (3:2) as the eluent yielded disaccharide saponins 5a and
5b.
Diosgenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4,6-di-O-
benzylidene-β-D-galactopyranoside (5a)[2]
This was prepared according to the above procedure from 660 mg of 7,
415 mg of diosgenin 8a, and 650 mg of 9 to get 638 mg of 5a (68%) as a white
foamy solid. [α]D20 –97.6 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.52–7.34
(m, 5 H), 5.54 (s, 1H, PhCH), 5.39 (d, 1H, J = 4.6 Hz, H-6), 5.35 (dd, 1H, J
= 3.4, 1.6 Hz, H-2Rha), 5.32 (d, 1H, J = 1.6 Hz, H-1Rha), 5.28 (dd, 1H, J =
10.0, 3.4 Hz, H-3Rha), 5.07 (t, 1H, J = 10.0 Hz, H-4Rha), 4.50 (m, 1H, H-5Rha),
4.48 (d, 1H, J = 7.2 Hz, H-1Gal), 4.41 (q, 1H, J = 7.4 Hz, H-16), 4.31 (d, 1H, J
= 11.9 Hz, H-6aGal), 4.15 (d, 1H, J = 3.5 Hz, H-4Gal), 4.07 (d, 1H, J = 11.9 Hz,
H-6bGal), 3.82 (dd, 1H, J = 9.6, 3.5 Hz, H-3Gal), 3.77 (dd, 1H, J = 9.6, 7.2 Hz,
H-2Gal), 3.63 (m, 1H, H-3), 3.42–3.51 (m, 2H, H-26a, H-5Gal), 3.37 (t, 1H, J =
10.9 Hz, H-26b), 2.12, 2.02, 1.98 (3s, 3 × 3 H, 3Ac), 1.21 (d, 3H, J = 6.2 Hz,
H-6Rha), 1.03 (s, 3H, CH3-19), 0.97 (d, 3H, J = 6.9 Hz, CH3-21), 0.79 (d, 3H,
J = 6.0 Hz, CH3-27), 0.78 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ
170.08, 170.06, 170.02, 140.4, 121.8, 109.3, 101.4, 98.9, 97.3, 80.8, 78.3, 75.7,
74.5, 74.2, 71.3, 69.6, 69.3, 69.2, 66.8, 66.4, 66.1, 62.1, 56.5, 50.1, 41.6, 40.3,
39.8, 38.4, 37.2, 36.9, 32.1, 31.8, 31.42, 31.39, 30.3, 29.5, 28.8, 20.95, 20.88,
20.85, 20.80, 19.3, 17.2, 17.1, 16.3, 14.5. ESI-HRMS (positive ion): Calcd for
(C52H72O15+NH4+): 954.5209; found m/z: 954.5222.

Indioside E Analogs and Their Antitumor Activities
159
Tigogenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4,6-di-O-
benzylidene-β-D-galactopyranoside (5b)
This was prepared according to the above procedure from 660 mg of 7,
418 mg of diosgenin 8b, and 650 mg of 9 to get 610 mg of 5b (65%) as a white
foamy solid. [α]D20 –84.4 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3): δ 7.52–7.34
(m, 5 H), 5.54 (s, 1H, PhCH), 5.38–5.27 (m, 3H, H-1,2,3Rha), 5.07 (t, 1H, J =
10.0 Hz, H-4Rha), 4.52 (m, 1H, H-5Rha), 4.48 (d, 1H, J = 7.2 Hz, H-1Gal), 4.39
(q, 1H, J = 7.4 Hz, H-16), 4.31 (d, 1H, J = 11.9 Hz, H-6aGal), 4.14 (d, 1H, J =
3.5 Hz, H-4Gal), 4.07 (d, 1H, J = 11.9 Hz, H-6bGal), 3.86–3.74 (m, 2H, H-2,3Gal),
3.63 (m, 1H, H-3), 3.48 (dd, 1H, J = 10.9, 3.2 Hz, H-26a), 3.45 (s, 1H, H-5Gal),
3.38 (t, 1H, J = 10.9 Hz, H-26b), 2.13, 2.04, 1.98 (3s, 3 × 3 H, 3Ac), 1.20
(d, 3H, J = 6.2 Hz, H-6Rha), 0.97 (d, 3H, J = 6.9 Hz, CH3-21), 0.83 (s, 3H,
CH3-19), 0.79 (d, 3H, J = 6.2 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13C NMR
(100 MHz, CDCl3): δ 170.03, 170.00, 169.97, 109.2, 101.3, 98.8, 97.2, 80.8, 77.9,
75.8, 74.2 (2C), 71.3, 69.7, 69.3, 69.1, 66.8, 66.4, 66.1, 62.2, 56.3, 54.3, 44.6,
41.6, 40.6, 40.0, 36.9, 35.8, 35.1, 34.2, 32.3, 31.8, 31.4, 30.3, 29.3, 28.8, 28.7,
21.0, 20.9, 20.8, 20.7, 17.2, 17.1, 16.5, 14.5, 12.2. ESI-HRMS (positive ion):
Calcd for (C52H74O15+NH4+): 956.5366; found m/z: 956.5380.
General Procedure for Preparation of Fully Protected Saponins
10, 11a–c, and 12a-c
To a solution of saponins 5a or 5b (0.15 mmol) and imidates 6a–d
(0.30 mmol) in anhydrous CH2Cl2 (5 mL) was added TMSOTf (9 μL,
0.05 mmol) at 0◦C under an N2 atmosphere. The reaction mixture was stirred
under this condition for 1 h, neutralized with Et3N, and concentrated to dry-
ness. The residue was subjected to flash chromatograph with petroleum ether
and EtOAc (3:1) as the eluent to give 10, 11a–c, and 12a–c.
Tigogenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
β-D-xylopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-galactopyranoside
(10)[3]
Prepared from 5b (140 mg) and 6a (182 mg) to yield 10 (147 mg, 71%) as
20
1
a white foamy solid. [α]D –73.4 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3):
δ 8.09–7.06 (m, 20H, Ph), 5.65 (t, 1H, J = 3.2 Hz, H-3Xyl), 5.54 (s, 1H, PhCH),
5.34–5.27 (m, 2H, H-3Rha, H-4Xyl), 5.26–5.20 (m, 3H, H-1,2Rha, H-1Xyl), 5.08 (d,
1H, J = 3.2 Hz, H-2Xyl), 4.97 (t, 1H, J = 10.0 Hz, H-4Rha), 4.71 (dd, 1H, J =
12.9, 1.8 Hz, H-5aXyl), 4.57 (d, 1H, J = 7.8 Hz, H-1Gal), 4.51 (m, 1H, H-5Rha),
4.46 (d, 1H, J = 3.5 Hz, H-4Gal), 4.39 (q, 1H, J = 7.4 Hz, H-16), 4.33 (d, 1H, J =
12.0 Hz, H-6aGal), 4.15–4.05 (m, 2H, H-2,6bGal), 3.89 (dd, 1H, J = 9.6, 3.5 Hz,
H-3Gal), 3.85 (d, 1H, J = 12.9 Hz, H-5bXyl), 3.71 (m, 1H, H-3), 3.52–3.42 (m,
2H, H-5Gal, H-26a), 3.37 (t, 1H, J = 10.9 Hz, H-26b), 2.00, 1.95, 1.88 (3s, 3 ×
3H, 3Ac), 1.09 (d, 3H, J = 6.2 Hz, H-6Rha), 0.96 (d, 3H, J = 6.9 Hz, CH3-21),

Y. Zhao et al.
160
0.83 (s, 3H, CH3-19), 0.79 (d, 3H, J = 6.1 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13
C
NMR (100 MHz, CDCl3): δ 170.1, 170.0, 169.9, 165.5, 165.1, 164.6, 109.3, 100.8,
100.0, 98.9, 97.0, 83.8, 80.8, 77.7, 76.0, 71.5, 71.5, 70.1, 69.3, 68.8, 68.3, 67.0,
66.8, 66.2 (2C), 65.9, 62.2, 58.7, 56.3, 54.3, 44.6, 41.6, 40.6, 40.0, 37.0, 35.8,
35.1, 34.0, 32.3, 31.8, 31.4, 30.3, 29.2, 28.8, 28.7, 21.0, 20.9, 20.8, 20.6, 17.2
(2C), 16.5, 14.5, 12.3. ESI-HRMS (positive ion): Calcd for (C78H94O22+Na+):
1405.6192; found m/z: 1405.6137.
Diosgenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
α-D-lyxopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-galactopyranoside
(11a)
This was prepared from 5a (140 mg) and 6b (182 mg) to yield 11a
20
1
(158 mg, 76%) as a white foamy solid. [α]D –69.6 (c 0.5, CHCl3); H NMR
(400 MHz, CDCl3): δ 8.13–7.26 (m, 20H, Ph), 5.75–5.66 (m, 3H, H-2,3,4Lyx),
5.49 (dd, 1H, J = 3.2, 1.6 Hz, H-2Rha), 5.44 (s, 1H, PhCH), 5.38 (d, 1H, J =
5.0 Hz, H-6), 5.36 (d, 1H, J = 1.2 Hz, H-1Lyx), 5.28–5.24 (m, 2H, H-1,3Rha),
5.12 (t, 1H, J = 10.0 Hz, H-4Rha), 4.64 (m, 1H, H-5Rha), 4.56 (d, 1H, J =
7.8 Hz, H-1Gal), 4.41 (q, 1H, J = 7.2 Hz, H-16), 4.37 (d, 1H, J = 3.2 Hz, H-
4Gal), 4.32 (d, 1H, J = 11.6 Hz, H-6aGal), 4.22–4.11 (m, 2H, H-2Gal, H-5aLyx),
4.06 (d, 1H, J = 11.6 Hz, H-6bGal), 3.99–3.88 (m, 2H, H-3Gal, H-5bLyx), 3.67
(m, 1H, H-3), 3.52–3.43 (m, 2H, H-5Gal, H-26a), 3.37 (t, 1H, J = 10.9 Hz, H-
26b), 2.02, 1.95, 1.94 (3s, 3 × 3H, 3Ac), 1.22 (d, 3H, J = 6.1 Hz, H-6Rha),
1.03 (s, 3H, CH3-19), 0.97 (d, 3H, J = 6.9 Hz, CH3-21), 0.79 (d, 3H, J =
6.0 Hz, CH3-27), 0.78 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ 170.1,
170.0, 169.9, 165.9, 165.1, 164.9, 140.5, 121.8, 109.3, 100.4, 98.7, 98.0, 95.0,
80.8, 77.6, 77.2, 72.1, 71.9, 71.4, 70.3, 69.7, 69.4, 69.3, 69.1, 67.2, 66.9, 66.2
(2C), 62.1, 61.5, 56.5, 50.1, 41.6, 40.3, 39.8, 38.2, 37.2, 36.9, 32.1, 31.9, 31.45,
31.40, 30.3, 29.4, 28.8, 20.9, 20.85, 20.82, 20.6, 19.3, 17.2, 17.1, 16.3, 14.5.
ESI-HRMS (positive ion): Calcd for (C78H92O22+NH4+): 1398.6419; found m/z:
1398.6448.
Diosgenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
β-D-ribopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-galactopyranoside
(11b)
Prepared from 5a (140 mg) and 6c (182 mg) to yield 11b (135 mg, 65%)
as a white foamy solid. [α]D –65.1 (c 0.5, CHCl3); 1H NMR (400 MHz,
20
CDCl3): δ 8.09–7.21 (m, 20H, Ph), 5.70 (d, 1H, J = 3.4 Hz, H-2Rib),
5.58 (t, 1H, J = 3.4 Hz, H-3Rib), 5.54 (m, 1H, H-4Rib), 5.51 (s, 1H,
PhCH), 5.39 (d, 1H, J = 4.9 Hz, H-6), 5.34 (s, 1H, H-1Rib), 5.32 (dd,
1H, J = 10.0, 3.2 Hz, H-3Rha), 5.25 (s, 1H, H-1Rha), 5.21 (dd, 1H, J =
3.2, 1.6 Hz, H-2Rha), 5.11 (t, 1H, J = 10.0 Hz, H-4Rha), 4.59 (m, 1H, H-5Rha),
4.58 (d, 1H, J = 7.7 Hz, H-1Gal), 4.48 (d, 1H, J = 12.4 Hz, H-5aRib), 4.43 (d, 1H,

Indioside E Analogs and Their Antitumor Activities
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J = 3.4 Hz, H-4Gal), 4.41 (q, 1H, J = 7.4 Hz, H-16), 4.29 (d, 1H, J = 11.6 Hz, H-
6aGal), 4.12 (dd, 1H, J = 9.6, 7.7 Hz, H-2Gal), 4.06 (d, 1H, J = 11.6 Hz, H-6bGal),
3.91 (dd, 1H, J = 9.6, 3.4 Hz, H-3Gal), 3.86 (d, 1H, J = 12.4 Hz, H-5bRib), 3.67 (m,
1H, H-3), 3.47 (dd, 1H, J = 10.9, 4.7 Hz, H-26a), 3.43 (s, 1H, H-5Gal), 3.38 (t, 1H,
J = 10.9 Hz, H-26b), 2.02, 1.95, 1.87 (3s, 3 × 3H, 3Ac), 1.24 (d, 3H, J = 6.1 Hz,
H-6Rha), 1.04 (s, 3H, CH3-19), 0.98 (d, 3H, J = 6.9 Hz, CH3-21), 0.79 (d, 3H,
J = 5.8 Hz, CH3-27), 0.79 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ 170.3,
170.0, 169.9, 166.2, 166.0, 165.1, 140.4, 121.9, 109.3, 101.3, 100.5, 98.8, 97.4,
84.1, 80.8, 77.8, 77.2, 75.3, 72.2, 71.6, 70.2, 69.3, 69.1, 68.9, 67.9, 66.8, 66.1,
65.7, 62.2, 62.1, 56.5, 50.1, 41.6, 40.3, 39.7, 38.2, 37.2, 36.9, 32.1, 31.8, 31.44,
31.39, 30.3, 29.4, 28.8, 20.87, 20.85, 20.81, 20.6, 19.3, 17.2, 17.1, 16.3, 14.5.
ESI-HRMS (positive ion): Calcd for (C78H92O22+NH4+): 1398.6419; found m/z:
1398.6445.
Diosgenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
α-L-arabinopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-
galactopyranoside (11c)
This was prepared from 5a (140 mg) and 6d (182 mg) to yield 11c (128 mg,
20
1
62%) as a white foamy solid. [α]D –49.9 (c 0.5, CHCl3); H NMR (400 MHz,
CDCl3): δ 8.10–7.00 (m, 20H, Ph), 5.85 (t, 1H, J = 3.4 Hz, H-2Ara), 5.65 (m, 1H,
H-4Ara), 5.59–5.53 (br s, 2H, H-3Ara, PhCH), 5.38 (d, 1H, J = 4.0 Hz, H-6), 5.28
(dd, 1H, J = 10.0, 3.4 Hz, H-3Rha), 5.23 (dd, 1H, J = 3.4, 1.0 Hz, H-2Rha), 5.17
(s, 1H, H-1Rha), 5.10 (s, 1H, H-1Ara), 4.95 (t, 1H, J = 10.0 Hz, H-4Rha), 4.64 (t,
1H, J = 10.6 Hz, H-5aAra), 4.56 (d, 1H, J = 7.7 Hz, H-1Gal), 4.51 (d, 1H, J =
3.2 Hz, H-4Gal), 4.46 (m, 1H, H-5Rha), 4.41 (q, 1H, J = 7.4 Hz, H-16), 4.33 (d,
1H, J = 12.2 Hz, H-6aGal), 4.15–4.02 (m, 2H, H-2,6bGal), 3.88 (dd, 1H, J =
9.6, 3.2 Hz, H-3Gal), 3.84 (dd, 1H, J = 10.6, 4.8 Hz, H-5bAra), 3.66 (m, 1H, H-
3), 3.51–3.43 (m, 2H, H-5Gal, H-26a), 3.38 (t, 1H, J = 10.9 Hz, H-26b), 1.99,
1.97, 1.94 (3s, 3 × 3H, 3Ac), 1.04 (d, 3H, J = 6.2 Hz, H-6Rha), 1.03 (s, 3H,
CH3-19), 0.97 (d, 3H, J = 7.0 Hz, CH3-21), 0.79 (d, 3H, J = 5.0 Hz, CH3-27),
0.78 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ 170.03 (2C), 169.99, 165.3,
165.2, 164.8, 140.4, 121.8, 109.3, 100.8, 99.7, 99.0, 97.1, 80.8, 78.0, 77.2, 76.1,
71.9, 71.5, 70.0, 69.9, 69.3, 68.9, 67.2, 66.8, 66.3, 65.9, 65.8, 65.6, 62.1, 56.5,
50.1, 41.6, 40.3, 39.8, 38.3, 37.2, 36.9, 32.1, 31.8, 31.4, 30.3, 29.7, 29.4, 28.8,
20.8, 20.6, 19.3, 17.14, 17.09, 16.3, 14.5. ESI-HRMS (positive ion): Calcd for
(C78H92O22+Na+): 1403.5972; found m/z: 1403.5985.
Tigogenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
α-D-lyxopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-galactopyranoside
(12a)
This was prepared from 5b (140 mg) and 6b (182 mg) to yield 12a (162 mg,
20
1
78%) as a white foamy solid. [α]D –54.2 (c 0.5, CHCl3); H NMR (400 MHz,

Y. Zhao et al.
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CDCl3): δ 8.14–7.25 (m, 20H, Ph), 5.78–5.63 (m, 3H, H-2,3Xyl, PhCH), 5.47
(br d, 1H, H-4Lyx), 5.44 (s, 1H, H-1Lyx), 5.36 (s, 1H, H-1Rha), 5.29 (dd, 1H,
H-3Rha), 5.27 (br s, 1H, H-2Rha), 5.08 (d, 1H, J = 2.6 Hz, H-2Xyl), 5.11 (t,
1H, J = 10.0 Hz, H-4Rha), 4.63 (m, 1H, H-5Rha), 4.56 (d, 1H, J = 7.7 Hz,
H-1Gal), 4.38 (q, 1H, J = 7.4 Hz, H-16), 4.37 (d, 1H, J = 3.6 Hz, H-4Gal),
4.33 (d, 1H, J = 12.3 Hz, H-6aGal), 4.23–4.10 (m, 2H, H-2Gal, H-5aLyx), 4.06
(d, 1H, J = 12.0 Hz, H-6bGal), 3.98–3.88 (m, 2H, H-3Gal, H-5bXyl), 3.71 (m,
1H, H-3), 3.50–3.41 (m, 2H, H-5Gal, H-26a), 3.37 (t, 1H, J = 10.9 Hz, H-
26b), 2.03, 1.95, 1.94 (3s, 3 × 3H, 3Ac), 1.21 (d, 3H, J = 6.1 Hz, H-6Rha),
0.96 (d, 3H, J = 6.9 Hz, CH3-21), 0.83 (s, 3H, CH3-19), 0.798 (d, 3H, J =
6.2 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ 170.1,
170.0, 169.9, 165.9, 165.1, 164.9, 109.3, 100.4, 98.7, 97.9, 94.9, 80.8, 77.3,
71.9 (2C), 71.4, 70.3, 69.6, 69.4, 69.3, 69.2, 68.2, 67.2, 66.8, 66.21, 66.16,
62.2, 61.5, 56.3, 54.3, 41.6, 40.6, 40.0, 37.0, 35.8, 35.1, 33.9, 32.3, 31.8, 31.4,
30.3, 29.2, 28.9, 28.8, 21.0, 20.9, 20.8, 20.7, 17.1 (2C), 16.5, 14.5, 12.3. ESI-
HRMS (positive ion): Calcd for (C78H94O22+NH4+): 1400.6575; found m/z:
1400.6590.
Tigogenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
β-D-ribopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-galactopyranoside
(12b)
This was prepared from 5b (140 mg) and 6c (182 mg) to yield 12b (126 mg,
20
1
61%) as a white foamy solid. [α]D –59.6 (c 0.5, CHCl3); H NMR (400 MHz,
CDCl3): δ 8.10–7.22 (m, 20H, Ph), 5.70 (d, 1H, J = 3.5 Hz, H-2Rib), 5.58 (t,
1H, J = 3.5 Hz, H-3Rib), 5.53 (m, 1H, H-4Rib), 5.51 (s, 1H, PhCH), 5.35 (dd,
1H, J = 10.0, 3.4 Hz, H-3Rha), 5.34 (s, 1H, H-1Rib), 5.25 (d, 1H, J = 1.5 Hz,
H-1Rha), 5.20 (dd, 1H, J = 3.4, 1.5 Hz, H-2Rha), 5.10 (t, 1H, J = 10.0 Hz,
H-4Rha), 4.61 (m, 1H, H-5Rha), 4.57 (d, 1H, J = 7.6 Hz, H-1Gal), 4.48 (d, 1H,
J = 12.2 Hz, H-5aRib), 4.42 (d, 1H, J = 3.0 Hz, H-4Gal), 4.39 (q, 1H, J =
7.4 Hz, H-16), 4.31 (d, 1H, J = 11.4 Hz, H-6aGal), 4.11 (dd, 1H, J = 9.6,
7.6 Hz, H-2Gal), 4.06 (d, 1H, J = 11.4 Hz, H-6bGal), 3.98 (dd, 1H, J = 9.6,
3.0 Hz, H-3Gal), 3.87 (d, 1H, J = 12.2 Hz, H-5bRib), 3.73 (m, 1H, H-3), 3.46
(dd, 1H, J = 10.9, 5.6 Hz, H-26a), 3.42 (s, 1H, H-5Gal), 3.37 (t, 1H, J =
10.9 Hz, H-26b), 2.03, 1.96, 1.87 (3s, 3 × 3H, 3Ac), 1.22 (d, 3H, J = 6.2 Hz,
H-6Rha), 0.96 (d, 3H, J = 6.9 Hz, CH3-21), 0.84 (s, 3H, CH3-19), 0.79 (d, 3H,
J = 6.2 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3): δ
170.3, 170.0, 169.9, 166.2, 166.0, 165.1, 109.2, 101.3, 100.5, 98.8, 97.3, 84.2,
80.8, 77.5, 75.3, 72.0, 71.7, 70.3, 69.3, 69.1, 68.8, 67.9, 66.8, 66.2, 66.1, 65.7,
62.3, 62.2, 56.3, 54.4, 44.6, 41.6, 40.6, 40.1, 37.0, 35.8, 35.1, 33.9, 32.3, 31.8,
31.4, 30.3, 29.3, 28.8, 28.8, 21.0, 20.9, 20.8, 20.6, 17.1, 16.5, 14.5, 12.3. ESI-
HRMS (positive ion): Calcd for (C78H94O22+NH4+): 1400.6575; found m/z:
1400.6603.

Indioside E Analogs and Their Antitumor Activities
163
Tigogenyl 2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4-tri-O-benzoyl-
α-L-arabinopyranosyl-(1→3)]-4,6-di-O-benzylidene-β-D-
galactopyranoside (12c)
This was prepared from 5b (140 mg) and 6d (182 mg) to yield 12c (137 mg,
20
1
66%) as a white foamy solid. [α]D –31.4 (c 0.5, CHCl3); H NMR (400 MHz,
CDCl3): δ 8.10–6.98 (m, 20H, Ph), 5.85 (t, 1H, J = 3.5 Hz, H-2Ara), 5.64 (m,
1H, H-4Ara), 5.58–5.53 (m, 2H, H-3Ara, PhCH), 5.30 (dd, 1H, J = 10.0, 3.4 Hz,
H-3Rha), 5.22 (dd, 1H, J = 3.4, 1.0 Hz, H-2Rha), 5.18 (s, 1H, H-1Rha), 5.10 (s, 1H,
H-1Ara), 4.94 (t, 1H, J = 10.0 Hz, H-4Rha), 4.65 (t, 1H, J = 10.5 Hz, H-5aAra),
4.56 (d, 1H, J = 7.8 Hz, H-1Gal), 4.51 (d, 1H, J = 3.5 Hz, H-4Gal), 4.46 (m,
1H, H-5Rha), 4.39 (q, 1H, J = 7.4 Hz, H-16), 4.34 (d, 1H, J = 13.1 Hz, H-6aGal),
4.13–4.04 (m, 2H, H-2,6bGal), 3.86 (dd, 1H, J = 9.6, 3.5 Hz, H-3Gal), 3.85 (dd, 1H,
J = 10.5, 4.6 Hz, H-5bAra), 3.71 (m, 1H, H-3), 3.51–3.43 (m, 2H, H-5Gal, H-26a),
3.37 (t, 1H, J = 10.9 Hz, H-26b), 1.99, 1.97, 1.94 (3s, 3 × 3H, 3Ac), 1.02 (d, 3H,
J = 6.2 Hz, H-6Rha), 0.96 (d, 3H, J = 6.8 Hz, CH3-21), 0.82 (s, 3H, CH3-19), 0.79
(d, 3H, J = 6.2 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13C NMR (100 MHz, CDCl3):
δ 170.1, 170.0, 169.9, 165.3, 165.2, 164.8, 109.3, 100.8, 99.7, 99.0, 96.9, 80.8,
77.8, 77.3, 76.2, 71.6, 70.1, 69.9, 69.3, 68.8, 67.1, 66.8, 66.3, 65.9, 65.8, 62.2,
56.3, 54.3, 44.6, 41.6, 40.6, 40.0, 37.0, 35.8, 35.1, 34.0, 32.3, 31.8, 31.4, 30.3,
29.2, 28.8, 28.7, 21.0, 20.9, 20.8, 20.7, 17.2, 17.1, 16.5, 14.5, 12.3. ESI-HRMS
(positive ion): Calcd for (C78H94O22+NH4+): 1400.6575; found m/z: 1400.6603.
General Procedure for Preparation of Target Molecules 2–4
A solution of fully protected saponins 10, 11a–c, or 12a–c (0.05 mmol) in
80% aq. AcOH (10 mL) was stirred at 70◦C for 3 h, when TLC (1:1 petroleum
ether-EtOAc) showed the completion of the reaction. The solvent was coevapo-
rated with toluene (3 × 15 mL) under reduced pressure to give a residue, which
was dissolved in methanol (10 mL). To this solution was added 1 M MeONa in
methanol until the pH value reached 10. The reaction mixture was stirred
at rt for 3 h and then neutralized with Amberlite IR 120 (H+). The mixture
was filtered off through filter paper, and the filtrate was combined and concen-
trated under diminished pressure. The residue was then subjected to column
chromatography with CH2Cl2-MeOH (3:1) as the eluent to afford the desired
saponins 2–4.
Tigogenyl α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-
galactopyranoside (2)[3]
This was prepared from 10 (70 mg) to yield 2 (40.3 mg, 93%) as a white
foamy solid. [α]D –74.0 (c 0.1, 2:3 CHCl3/CH3OH); 1H NMR (600 MHz,
20
2:1 CDCl3/CD3OD): δ 5.16 (s, 1H, H-1Rha), 4.46 (d, 1H, J = 7.8 Hz, H-1Gal),
4.43–4.37 (m, 2H, H-1Xyl, H-16), 4.10 (m, 1H, H-5Rha), 4.09 (s, 1H), 4.03 (d, 1H,
J = 2.4 Hz), 3.95 (br s, 1H), 3.91 (dd, 1H, J = 11.5, 5.1 Hz, H-5aXyl), 3.81–3.74

Y. Zhao et al.
164
(m, 3H), 3.73–3.61 (m, 3H), 3.55 (m 1H), 3.51–3.44 (m, 2H), 3.39 (t, 1H, J =
9.5 Hz), 3.36 (m, 1H), 3.31 (dd, 1H, J = 8.5, 7.6 Hz), 3.23 (t, 1H, J = 10.7 Hz, H-
26b), 1.26 (d, 3H, J = 6.1 Hz, H-6Rha), 0.97 (d, 3H, J = 6.9 Hz, CH3-21), 0.83 (s,
3H, CH3-19), 0.80 (d, 3H, J = 6.2 Hz, CH3-27), 0.77 (s, 3H, CH3-18). 13C NMR
(150 MHz, 2:1 CDCl3/CD3OD ): δ 109.5, 104.7 (C-1Xyl), 100.8 (C-1Rha), 99.1 (C-
1Gal), 83.5, 80.9, 76.0, 74.5, 73.9, 73.0, 72.6, 71.0, 70.4, 69.4, 69.1, 68.3, 66.7,
65.4, 61.9, 61.1, 56.2, 54.3, 44.5, 41.5, 40.5, 39.9, 36.9, 35.6, 34.9, 33.6, 32.2,
31.4, 31.1, 30.1, 29.1, 28.6, 28.5, 20.9, 17.1, 16.8, 16.3, 14.1, 11.9. ESI-HRMS
(positive ion): Calcd for (C44H72O16+NH4+): 874.5159; found m/z: 874.5165.
Diosgenyl α-L-rhamnopyranosyl-(1→2)-[α-D-lyxopyranosyl-(1→3)]-β-D-
galactopyranoside (3a)
This was prepared from 11a (70 mg) to yield 3a (40.7 mg, 94%) as a
white foamy solid. [α]D20 –43.0 (c 0.1, 2:3 CHCl3/CH3OH); 1H NMR (600 MHz,
CD3OD): δ 5.37 (d, 1H, J = 4.3 Hz, H-6), 5.10 (s, 1H, H-1Rha), 4.76 (d, 1H, H-
1Lyx, overlapped by CD3OD), 4.48 (t, 1H, J = 3.6 Hz), 4.38 (q, 1H, J = 7.2 Hz,
H-16), 4.15 (m, 1H, H-5Rha), 4.05 (s, 1H), 3.94 (br s, 1H), 3.80–3.73 (m, 6H),
3.73–3.69 (m, 2H), 3.64 (dd, 1H, J = 9.6, 3.6 Hz), 3.61 (m 1H, H-3), 3.57 (dd
1H, J = 10.2, 4.2 Hz), 3.47 (t, 1H, J = 6.2 Hz), 3.43 (dd, 1H, J = 10.9, 2.7 Hz,
H-26a), 3.37 (t, 1H, J = 9.5 Hz), 3.31 (t, 1H, J = 10.9 Hz, H-26b), 1.22 (d, 3H,
J = 6.2 Hz, H-6Rha), 1.03 (s, 3H, CH3-19), 0.95 (d, 3H, J = 7.0 Hz, CH3-21),
0.80 (s, 3H, CH3-18), 0.78 (d, 3H, J = 5.4 Hz, CH3-27). 13C NMR (150 MHz,
2:1 CDCl3/CD3OD): δ 140.4, 121.5, 109.6, 100.8 (C-1Rha), 99.5 (C-1Gal), 97.1 (C-
1Lyx), 80.9, 78.7, 78.1, 74.0, 73.2, 72.6, 71.0 (2C), 70.1, 69.2, 68.4, 68.1, 66.8,
64.9, 64.1, 61.8, 61.1, 56.4, 50.0, 41.5, 40.2, 39.6, 38.2, 37.1, 36.7, 31.9, 31.5,
31.3, 31.1, 30.1, 29.4, 28.5, 20.7, 19.0, 17.0, 16.8, 16.1, 14.2. ESI-HRMS (posi-
tive ion): Calcd for (C44H70O16+NH4+): 872.5002; found m/z: 872.5012.
Diosgenyl α-L-rhamnopyranosyl-(1→2)-[β-D-ribopyranosyl-(1→3)]-β-D-
galactopyranoside (3b)
Prepared from 11b (70 mg) to yield 3b (39.8 mg, 92%) as a white
20
1
foamy solid. [α]D –113.0 (c 0.1, 2:3 CHCl3/CH3OH); H NMR (600 MHz, 2:1
CDCl3/CD3OD): δ 5.37 (d, 1H, J = 4.6 Hz, H-6), 5.07 (s, 1H, H-1Rha), 4.92 (d,
1H, J = 3.9 Hz, H-1Rib), 4.47 (d, 1H, J = 7.7 Hz, H-1Gal), 4.42 (q, 1H, J = 7.2 Hz,
H-16), 4.11 (m, 1H, H-5Rha), 4.02 (d, 1H, J = 2.8 Hz), 3.98–3.92 (m, 2H), 3.89
(br s, 1H), 3.83–3.74 (m, 4H), 3.71 (dd, 1H, J = 11.7, 5.3 Hz), 3.69–3.61 (m, 4H),
3.52–3.45 (m, 2H), 3.61 (m, 1H, H-3), 3.40 (t, 1H, J = 9.6 Hz), 3.36 (t, 1H, J =
11.4 Hz, H-26b), 1.27 (d, 3H, J = 6.2 Hz, H-6Rha), 1.04 (s, 3H, CH3-19), 0.98 (d,
3H, J = 7.0 Hz, CH3-21), 0.81 (s, 3H, CH3-18), 0.80 (d, 3H, J = 6.5 Hz, CH3-27).
13C NMR (150 MHz, 2:1 CDCl3/CD3OD): δ 140.4, 121.6, 109.6, 102.8 (C-1Rib),
100.9 (C-1Rha), 99.6 (C-1Gal), 82.8, 80.9, 78.0, 74.9, 74.1, 72.5, 71.0, 70.8, 70.4,
69.0, 68.7, 68.4, 67.0, 66.8, 64.0, 61.8, 61.1, 56.4, 50.0, 41.5, 40.2, 39.6, 38.1,
37.1, 36.7, 31.9, 31.5, 31.3, 31.1, 30.1, 29.4, 28.5, 20.7, 19.0, 17.1, 16.8, 16.1,

Indioside E Analogs and Their Antitumor Activities
165
14.2. ESI-HRMS (positive ion): Calcd for (C44H70O16+NH4+): 872.5002; found
m/z: 872.5016.
Diosgenyl α-L-rhamnopyranosyl-[(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-
galactopyranoside (3c)
This was prepared from 11c (70 mg) to yield 3c (40.7 mg, 94%) as a white
foamy solid. [α]D –109.0 (c 0.1, 2:3 CHCl3/CH3OH); 1H NMR (600 MHz,
20
CD3OD): δ 5.38 (d, 1H, J = 4.8 Hz, H-6), 5.18 (s, 1H, H-1Rha), 4.47 (d, 1H, J
= 7.8 Hz, H-1Gal), 4.40 (d, 1H, J = 7.0 Hz, H-1Ara), 4.39 (q, 1H, J = 7.2 Hz,
H-16), 4.13 (m, 1H, H-5Rha), 4.03 (d, 1H, J = 2.9 Hz), 3.91 (br s, 1H), 3.84 (dd,
1H, J = 12.4, 2.8 Hz), 3.82–3.79 (m, 1H), 3.76 (dd, 1H, J = 9.4, 7.8 Hz, H-2Gal),
3.71–3.66 (m, 4H), 3.63 (dd, 1H, J = 9.4, 3.4 Hz), 3.61 (m, 1H, H-3), 3.60 (dd,
1H, J = 9.0, 7.0 Hz, H-2Ara), 3.54 (d,1H, J = 11.9 Hz), 3.52–3.47 (m, 2H), 3.43
(m, 1H), 3.37 (t, 1H, J = 9.5 Hz), 3.31 (t, 1H, J = 10.9 Hz, H-26b), 1.22 (d, 3H,
J = 6.2 Hz, H-6Rha), 1.03 (s, 3H, CH3-19), 0.95 (d, 3H, J = 7.0 Hz, CH3-21),
0.79 (s, 3H, CH3-18), 0.78 (d, 3H, J = 6.4 Hz, CH3-27). 13C NMR (150 MHz,
2:1 CDCl3/CD3OD): δ 140.4, 121.5, 109.6, 104.8 (C-1Ara), 100.6 (C-1Rha), 99.4
(C-1Gal), 83.7, 80.9, 77.9, 74.4, 73.9, 71.1, 71.0, 70.4, 68.9, 68.3, 68.1, 66.8, 65.7,
61.8, 61.1, 56.4, 50.0, 41.5, 40.2, 39.6, 38.1, 37.1, 36.8, 31.9, 31.5, 31.3, 31.1,
30.1, 29.5, 28.5, 20.7, 19.0, 17.1, 16.8, 16.1, 14.2. ESI-HRMS (positive ion):
Calcd for (C44H70O16+NH4+): 872.5002; found m/z: 872.5012.
Tigogenyl α-L-rhamnopyranosyl-(1→2)-[α-D-lyxopyranosyl-(1→3)]-β-D-
galactopyranoside (4a)
This was prepared from 12a (70 mg) to yield 4a (41.6 mg, 96%) as a
white foamy solid. [α]D20 –43.0 (c 0.1, 2:3 CHCl3/CH3OH); 1H NMR (600 MHz,
CD3OD): δ 5.08 (s, 1H, H-1Rha), 4.78 (d, 1H, H-1Lyx, overlapped by CD3OD),
4.48 (d, 1H, J = 6.0 Hz, H-1Gal), 4.36 (q, 1H, J = 7.0 Hz, H-16), 4.13 (m, 1H,
H-5Rha), 4.05 (s, 1H), 3.94 (br s, 1H), 3.80–3.67 (m, 8H), 3.63 (dd, 1H, J = 9.0,
3.0 Hz), 3.57 (dd, 1H, J = 9.6, 3.0 Hz), 3.46 (t, 1H, J = 6.0 Hz), 3.43 (dd, 1H,
J = 10.9, 3.6 Hz, H-26a), 3.37 (t, 1H, J = 9.5 Hz), 3.30 (t, 1H, J = 10.9 Hz,
H-26b), 1.20 (d, 3H, J = 6.1 Hz, H-6Rha), 0.93 (d, 3H, J = 7.0 Hz, CH3-21), 0.84
(s, 3H, CH3-19), 0.77 (d, 3H, J = 6.7 Hz, CH3-27), 0.76 (s, 3H, CH3-18). 13C
NMR (150 MHz, 2:1 CDCl3/CD3OD): δ 109.5, 100.9 (C-1Rha), 99.3 (C-1Gal), 97.1
(C-1Lyx), 80.9, 78.8, 73.9, 73.3, 72.6, 71.0 (2C), 70.1, 69.2, 68.3, 68.1, 66.8, 65.0,
64.2, 61.9, 61.1, 56.2, 54.3, 44.5, 41.5, 40.5, 39.9, 36.9, 35.6, 34.9, 33.7, 32.1,
31.5, 31.1, 30.1, 29.1, 28.5 (2C), 20.9, 17.0, 16.8, 16.3, 14.1, 11.9. ESI-HRMS
(positive ion): Calcd for (C44H72O16+NH4+): 874.5159; found m/z: 874.5168.
Tigogenyl α-L-rhamnopyranosyl-(1→2)-[β-D-ribopyranosyl-(1→3)]-β-D-
galactopyranoside (4b)
This was prepared from 12b (70 mg) to yield 4b (41.2 mg, 94%) as a white
20
1
foamy solid. [α]D –92.0 (c 0.1, 2:3 CHCl3/CH3OH); H NMR (600 MHz, 3:1

Y. Zhao et al.
166
CDCl3/CD3OD): δ 5.05 (s, 1H, H-1Rha), 4.92 (d, 1H, J = 3.9 Hz, H-1Rib), 4.48 (d,
1H, J = 7.7 Hz, H-1Gal), 4.39 (q, 1H, J = 7.4 Hz, H-16), 4.10 (m, 1H, H-5Rha),
4.01 (d, 1H, J = 2.9 Hz), 3.98–3.92 (m, 2H), 3.88 (dd, 1H, J = 3.0, 1.4 Hz),
3.83–3.72 (m, 5H), 3.70 (dd, 1H, J = 11.7, 5.2 Hz), 3.67–3.62 (m, 3H), 3.52–3.45
(m, 2H), 3.39 (t, 1H, J = 9.6 Hz), 3.35 (t, 1H, J = 11.4 Hz, H-26b), 1.27 (d, 3H,
J = 6.2 Hz, H-6Rha), 1.04 (s, 3H, CH3-19), 0.98 (d, 3H, J = 7.0 Hz, CH3-21),
0.81 (s, 3H, CH3-18), 0.81 (d, 3H, J = 6.6 Hz, CH3-27). 13C NMR (150 MHz,
2:1 CDCl3/CD3OD): δ 109.5, 102.9 (C-1Rib), 100.9 (C-1Rha), 99.2 (C-1Gal), 82.8,
80.9, 74.9, 74.1, 72.6, 71.1, 70.8, 70.4, 69.1, 68.8, 68.4, 67.0, 66.8, 64.0, 61.9,
61.2, 56.2, 54.3, 44.5, 41.5, 40.5, 39.9, 36.9, 35.6, 34.9, 33.6, 32.2, 31.5, 31.1,
30.1, 29.1, 28.6, 28.5, 20.9, 17.1, 16.8, 16.3, 14.1, 11.9. ESI-HRMS (positive
ion): Calcd for (C44H72O16+NH4+): 874.5159; found m/z: 874.5169.
Tigogenyl α-L-rhamnopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-β-D-
galacto pyranoside (4c)
Prepared from 12c (70 mg) to yield 4c (39.9 mg, 92%) as a white
20
1
foamy solid. [α]D –69.0 (c 0.1, 2:3 CHCl3/CH3OH); H NMR (600 MHz, 3:1
CDCl3/CD3OD): δ 5.17 (s, 1H, H-1Rha), 4.47 (d, 1H, J = 7.8 Hz, H-1Gal), 4.40
(d, 1H, J = 7.0 Hz, H-1Ara), 4.39 (q, 1H, J = 7.2 Hz, H-16), 4.12 (m, 1H, H-
5Rha), 4.06 (d, 1H, J = 2.9 Hz), 3.91 (dd, 1H, J = 3.2, 1.6 Hz), 3.88 (dd, 1H, J
= 12.4, 2.7 Hz), 3.85 (m, 1H), 3.78 (dd, 1H, J = 9.4, 7.8 Hz, H-2Gal), 3.76–3.69
(m, 4H), 3.68–3.63 (m, 2H), 3.61 (dd, 1H, J = 9.0, 7.0 Hz, H-2Ara), 3.55 (d, 1H,
J = 11.9 Hz), 3.53 (dd, 1H, J = 9.1, 3.3 Hz), 3.49 (t, 1H, J = 6.0 Hz), 3.46 (m,
1H, H-26a), 3.40 (t, 1H, J = 9.6 Hz), 3.33 (t, 1H, J = 10.9 Hz, H-26b), 1.24 (d,
3H, J = 6.2 Hz, H-6Rha), 0.96 (d, 3H, J = 7.0 Hz, CH3-21), 0.85 (s, 3H, CH3-19),
0.80 (d, 3H, J = 6.3 Hz, CH3-27), 0.78 (s, 3H, CH3-18). 13C NMR (150 MHz,
2:1 CDCl3/CD3OD): δ 109.5, 104.8 (C-1Ara), 100.7 (C-1Rha), 99.1 (C-1Gal), 83.7,
80.9, 74.5, 73.9, 72.7, 72.6, 71.1, 71.0, 70.4, 69.0, 68.3, 68.1, 66.8, 65.7, 61.9,
61.2, 56.2, 54.3, 44.5, 41.5, 40.5, 39.9, 36.9, 35.6, 34.9, 33.6, 32.2, 31.4, 31.1,
30.1, 29.1, 28.6, 28.5, 20.9, 17.0, 16.8, 16.3, 14.2, 11.9. ESI-HRMS (positive
ion): Calcd for (C44H72O16+NH4+): 874.5159; found m/z: 874.5176.
Cell Culture Assays
BEL-7402 and MCF-7 cells were grown in GIBCO RPMI Media 1640 sup-
plemented with 10% fetal bovine serum. All cells were incubated at 37◦C un-
der 5% CO2. The antiproliferative activities of saponins 1–4 were assessed
by means of the Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories,
Kumamoto, Japan), according to the manufacturer’s protocol. Briefly, 96-well
plates were seeded with 2 × 104 cells/mL and incubated overnight to allow
cells to attach to the plate surface. Cells were then exposed to serially diluted
solutions (0–40 μM) of 1–4 (to get a final volume of 100 μL) for 48 h. Then,
10 μL of CCK-8 was added into each well. After incubation at 37◦C for 3 h, the

Indioside E Analogs and Their Antitumor Activities
167
absorbance value (OD value) at 450 nm was determined by a PE EnSpire 2300
multimode reader. The rate of cell growth inhibition was calculated according
to the following equation:
Cell growth inhibition rate = [1 − (ODtreated − ODblank)/
(ODuntreated − ODblank)] × 100%
The IC50 value was determined by a modified Kou-type method:[17]
lgIC50= Xm − I[P − (3 − Pm − Pn)/4],
(1)
where Xm is the logarithmic maximum concentration, I is the logarithmic max-
imum concentration/adjacent concentration, P is the sum of the positive re-
sponse rate, Pm is the largest positive response rate, and Pn is the smallest
positive response rate. Each sample was tested in triplicate.
FUNDING
This work was supported by grants from the Major State Basic Research De-
velopment Program of China (973 Program, No. 2012CB822102), the National
Major Scientific and Technological Special Project for Significant New Drugs
Development (No. 2012ZX09502001-005), and the Shandong Provincial Natu-
ral Science Foundation (No. ZR2010BQ004).
SUPPLEMENTARY DATA
1
Supplementary data including H NMR, 13C NMR, and ESI-MS spectra of 5,
10–12, and 2–4 are available from the corresponding author.
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