The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22

Article abstract of DOI:10.1016/j.steroids.2010.07.011

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3β,22,26-trihyd

Full text of DOI:10.1016/j.steroids.2010.07.011

Steroids 76 (2011) 18–27
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Steroids
journal homepage: www.elsevier.com/locate/steroids
The synthesis of cholestane and furostan saponin analogues and the
determination of sapogenin’s absolute configuration at C-22
Haixing Wang, Yanshen Guo, Yuyao Guan, Liang Zhou, Pingsheng Lei∗
Key Laboratory of Bioactivity Substance and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 April 2010
Received in revised form 16 July 2010
Accepted 26 July 2010
Available online 20 August 2010
A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was
established and adopted for the first time. Following this strategy, starting from diosgenin, three
novel cholestane saponin analogues: (22S,25R)-3ˇ,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-˛-
l-rhamnopyranosyl-(1 → 2)-ˇ-d-glucopyranoside] 11, (25R)-3ˇ,16ˇ,26-trihydroxy-cholest-5-ene-22-
one 16-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-˛-d-glucopyranoside] 14 and (25R)-3ˇ,16ˇ,26-trihydroxy-
cholest-5-ene-22-one 16-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-ˇ-d-glucopyranoside] 17, three novel
furostan saponin analogues: (22S,25R)-furost-5-ene-3ˇ,22,26-triol 22-O-(˛-d-glucopyranoside) 23,
(22R,25R)-furost-5-ene-3ˇ,22,26-triol 22-O-(˛-d-glucopyranoside) 24 and (22S,25R)-furost-5-ene-
3ˇ,22,26-triol 22-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-˛-d-glucopyranoside] 26, were synthesized ulti-
mately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The
S-chirality at C-22 of cholestane was confirmed by Mosher’s method. The absolute configuration at C-22
of furostan saponin analogues was distinguished by conformational analysis combined with the NMR
spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown
also.
Keywords:
Cholestane
Furostan
Glycosylation
Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction
The research results concerning the synthesis and cytotoxi-
city estimation of icogenin analogues were published recently
The family of steroid saponins is an excellent source of
diverse chemical structures with a broad range of promising
pharmaceutical properties (e.g. antiviral, antibacterial, antifungal,
anti-inflammatory and antitumor) [1–6], which has been reported
in literature. According to the structure of sapogenin, steroid
saponins were divided into spirostan saponins, cholestane saponins
and furostan saponins. In 1992, the discovery of OSW-1 [7], a valu-
able cholestane saponin, attracted attention to the cytotoxicity
of this kind of compounds. Along with the development of phy-
tochemistry, many novel structured furostan saponins, just like
icogenin [8] and protodioscin [9] which showed good cytotoxic
activities, were separated and distinguished from nature plants.
Biological activity and bioavailability of steroid saponins did not
only depend on the structure of sapogenin, but also the saccha-
ride part, which decreased hydrophobic property of the steroidal
sapogenin and made it more available for biochemical processes in
the organism.
by our research group [10]. For further study, more cholestane
and furostan saponin analogues were designed and synthe-
sized. One of the intermediates, (25R)-26-acetyloxy-3ˇ-tert-
butyldimethylsilyloxycholest-5-ene-16,22-dione 1 [10], was pre-
pared using the most common steroid sapogenin diosgenin as raw
material. This compound was taken as the starting material for the
synthesis of target saponin analogues.
2. Experimental
2.1. General
Optical rotatings were recorded in a Perkin-Elmer 341 LC
polarimeter. 1H NMR and 13C NMR spectra were recorded on Varian
Unity Inova 300, 400, 500 and 600 MHz spectrometers. The chem-
ical shifts are reported in ppm using TMS as an internal standard.
Mass spectra were obtained on a VGZAB-2F mass spectrometer for
ESI-MS. HRMS was recorded on an Aglient 1100 series LC/MSD TOF.
Analytical thin layer chromatography (TLC) was carried out on TLC
plates silica gel 60 F254 percolated by Branch of Qingdao Haiyang
Chemical Plant. Chromatography was performed with silica gel H
(HG/T2354-92) and sephadex LH-20 (GE Healthcare).
∗
Corresponding author. Tel.: +86 10 63162006; fax: +86 10 63017757.
E-mail addresses: wangdizhu007@163.com (H. Wang), lei@imm.ac.cn (P. Lei).
0039-128X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.07.011

H. Wang et al. / Steroids 76 (2011) 18–27
19
2.2. Synthesis
at −15 ◦C for 15 min, then AgOTf (680 mg, 2.65 mmol) was added.
The reaction was stirred for 10 min avoiding the light, then 2.5 ml
distilled water was added and warmed up to room temperature.
After the reaction was stirred for 1 h, saturated aqueous NaHCO3
was added. The organic layer was separated and the water layer
was extracted with CH2Cl2 three times. The combined organic
layer was washed with brine and dried with anhydrous NaSO4,
and then filtered. The filtrate was concentrated in vacuo to give
a residue (1.09 g). The residue was carefully dried, dissolved in
dry CH2Cl2 (50 ml) under argon at 0 ◦C (ice bath), trichloroacetoni-
trile (0.56 ml, 5.65 mmol) and DBU (84 ␮l, 0.56 mmol) were added.
The reaction was stirred for 6 h, concentrated and the residue was
purified by column chromatography to yield compound 5 (860 mg,
67%).
2.2.1. (22R,25R)-26-Acetyloxy-3ˇ-tert-butyldimethylsilyloxy-
furost-5-ene-22-ol (compound 2) and (22S,25R)-26-acetyloxy-
3ˇ-tert-butyldimethylsilyloxy-cholest-5-ene-16ˇ,22-diol
(compound 3)
To a solution of compound 1 (2.50 g, 4.26 mmol) in dry THF
(60 ml) under argon cooled to −15 ◦C, NaBH4 (1.78 g, 46.86 mmol)
and CeCl3·7H2O (2.22 g, 5.96 mmol) were added. The reaction mix-
ture was stirred for 1 h, then cooled to −40 ◦C and quenched
with methyl alcohol. The suspension was diluted with CH2Cl2. The
organic layer was washed with saturated NaHCO3 and brine respec-
tively, and then dried over anhydrous Na2SO4, and filtered. The fil-
trate was concentrated in vacuo to give a residue, which was chro-
matographed on a silica gel column to afford compound 2 (899 mg,
1H NMR (ı, CDCl3, 300 MHz): 6.45 (d, 1H, J = 3.6 Hz, H-1), 5.51
(t, 1H, J = 9.6 Hz, H-4), 5.19 (dd, 1H, J = 3.3, 9.9 Hz, H-3ꢀ), 5.11 (t,
1H, J = 9.9 Hz, H-4ꢀ), 5.01 (dd, 1H, J = 1.8, 3.3 Hz, H-2ꢀ), 4.90 (d, 1H,
J = 1.5 Hz, H-1ꢀ), 4.29 (dd, 1H, J = 4.2, 12.3 Hz, H-6a), 4.15–4.19 (m,
1H, H-5), 4.07–4.12 (m, 1H, H-6b), 3.98 (dd, 1H, J = 3.6, 9.9 Hz, H-2),
3.83–3.88 (m, 1H, H-5ꢀ), 2.13 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.06 (s,
3H, Ac), 2.03 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.16 (d,
3H, J = 6.3 Hz, H-6ꢀ). 13C NMR (ı, CDCl3, 125 MHz): 170.47, 170.03
(2C), 169.90, 169.63, 169.51, 161.19 (C = NH), 94.16, 90.64, 76.11,
71.31, 70.86, 70.03 (3C), 68.05, 67.71, 67.28, 61.39, 20.83, 20.71,
20.62 (2C), 20.60, 20.52, 17.27. LC-HRESIMS m/z 744.0854 [M+Na]+
(calcd for C26H34O16NNaCl3 744.0835).
36%, white solid) and compound 3 (1.01 g, 42%, white solid).
Compound 2: m.p.: 104.2–106.2 ◦C. [˛]D
−51.5◦ (c = 0.66,
20
CH2Cl2). 1H NMR (ı, CDCl3, 300 MHz): 5.39 (d, 1H, J = 4.8 Hz, H-6),
4.59 (q, 1H, J = 6.9, 14.4 Hz, H-16), 3.81–4.01 (m, 2H, H-26), 3.47 (m,
1H, H-3), 2.12–2.30 (m, 2H), 2.06 (s, 3H, Ac), 0.05 (s, 6H). 13C NMR
(ı, d-pyridine, 100 MHz): 170.75 (Ac), 141.50 (C-5), 121.43 (C-6),
110.47 (C-22), 81.15 (C-16), 72.82 (C-3), 69.37 (C-26), 63.81, 56.68,
50.41, 43.34, 40.85, 40.68, 40.01, 37.49, 37.06, 36.92, 33.30, 32.56,
32.47, 32.38, 31.74, 28.21, 26.09 (t-Bu), 21.19, 20.74, 19.50, 18.32,
17.03, 16.51, 16.42, −4.35 ((CH3)2-Si). LC-HRESIMS m/z 589.4108
[M+H]+ (calcd for C35H61O5Si, 589.4163).
Compound 3: m.p.: 86.6–88.6 ◦C. 1H NMR (ı, CDCl3, 300 MHz):
5.31 (d, 1H, J = 4.2 Hz, H-6), 4.33 (dd, 1H, J = 3.9, 11.1 Hz, H-16),
3.83–4.00 (m, 2H), 3.61 (d, 1H, J = 9.3 Hz, H-22), 3.43–3.50 (m, 1H, H-
3), 2.05 (s, 3H, Ac), 0.04 (s, 6H, Me2Si). 13C NMR (ı, CDCl3, 100 MHz):
171.40 (Ac), 141.59 (C-5), 120.93 (C-6), 77.46 (C-22), 72.56 (C-3),
71.91 (C-16), 69.12 (C-26), 57.17 (C-17), 54.65 (C-14), 50.15 (C-9),
42.74, 42.47, 39.99, 37.30, 36.55, 35.95, 34.90, 32.65, 32.02, 31.80,
31.52, 30.71, 29.61, 25.91 (3C), 20.96, 20.71, 19.39, 18.22, 17.07,
16.01, 12.96, −4.61 (2C). LC-HRESIMS m/z 591.4394 [M+Na]+ (calcd
for C35H63O5Si 591.4439).
2.2.5. (22S,25R)-26-Acetyloxy-3ˇ-tert-
butyldimethylsilyloxycholest-5-ene-16ˇ,22-diol
22-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-ˇ-d-glucopyranoside] (compound 6),
(22S,25R)-26-acetyloxy-3ˇ-tert-butyldimethylsilyloxycholest-5-
ene-16ˇ,22-diol
16-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-˛-d-glucopyranoside] (compound 7) and
(22S,25R)-26-acetyloxy-3ˇ-tert-butyldimethylsilyloxycholest-5-
ene-16ˇ,22-diol
2.2.2. (S)-MTPA ester 3a
16-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-ˇ-d-glucopyranoside] (compound
8)
A solution of carefully dried cholestane sapogenin 3 (1.23 g,
1.70 mmol) and carefully dried disaccharide trichloroacetimidate
To a mixture of compound 3 (20 mg, 0.034 mmol), DMAP
(8.3 mg, 0.068 mmol) and triethylamine (7 ␮l, 0.051 mmol) in dry
CH2Cl2 (2 ml) was added (R) – MTPACl (13 ␮l, 0.068 mmol). The
reaction mixture was stirred at room temperature for 1 h, concen-
trated and the residue was purified by column chromatography to
yield the (S)-MTPA ester (3a) (20 mg, 74%).
˚
5 (2.01 g, 3.40 mmol) in dry CH2Cl2 (80 ml) was stirred with 4 A
molecular sieves (1.5 g) under argon at room temperature for
15 min, and then TMSOTf (31 ␮l, 0.17 mmol) was slowly added. The
reaction mixture was stirred for an additional 30 min, quenched
with triethylamine, diluted with CH2Cl2. The molecular sieves were
filtered out. The filtrate was washed with saturated NaHCO3 and
brine respectively, and then dried over anhydrous NaSO4, and fil-
tered. The filtrate was concentrated in vacuo to give a residue,
which was chromatographed on a silica gel column to afford three
products: compound 6 (190 mg, 10%), compound 7 (901 mg, 46%),
compound 8 (233 mg, 13%).
1H NMR (ı, CDCl3, 300 MHz): 7.558–7.583 (m, 2H, Ph),
7.393–7.410 (m, 3H, Ph), 5.457 (t, 1H, J = 6.9 Hz, H-22), 5.316 (d,
1H, J = 4.5 Hz, H-6), 4.390 (m, 1H, H-16), 3.713–3.971 (m, 2H, H-26),
3.509 (s, 3H, MeO), 3.470 (m, H-3), 2.029 (s, 3H, Ac), 0.997 (s, 3H,
H-19), 0.937 (d, 3H, J = 7.2 Hz, H-21), 0.909 (s, 3H, H-18), 0.860 (s,
3H, H-26), 0.060 (s, 6H, Me2Si).
2.2.3. (R)-MTPA ester 3b
Following the same procedure as 3a, compound 3 (20 mg,
0.034 mmol) was converted to (R)-MTPA ester (3b) (18 mg, 67%).
1H NMR (ı, CDCl3, 300 MHz): 7.567–7.591 (m, 2H, Ph),
7.383–7.420 (m, 3H, Ph), 5.427 (t, 1H, J = 6.9 Hz, H-22), 5.315 (d,
1H, J = 4.5 Hz, H-6), 4.319 (m, 1H, H-16), 3.747–3.977 (m, 2H, H-26),
3.570 (s, 3H, MeO), 3.456 (m, H-3), 2.030 (s, 3H, Ac), 0.984 (s, 3H,
H-19), 0.892 (s, 3H, H-26), 0.864 (d, 3H, J = 6.6 Hz, H-21), 0.853 (s,
3H, H-18), 0.054 (s, 6H, Me2Si).
Compound 6: 1H NMR (ı, CDCl3, 300 MHz): 5.22–5.29 (m, 3H,
H-6, 3ꢀ, 3ꢀꢀ), 5.07 (d, 1H, J = 1.5 Hz, H-1ꢀꢀ), 4.91–5.06 (m, 3H), 4.66 (d,
1H, J = 7.8 Hz, H-1ꢀ), 4.26–4.31 (m, 2H, H-16, 5ꢀꢀ), 3.99–4.16 (m, 3H),
3.84–3.94 (m, 2H, H-26), 3.65–3.75 (m, 2H), 3.42–3.52 (m, 1H, H-3),
2.10 (s, 3H), 2.06 (s, 3H), 2.03 (s, 6H), 2.01 (s, 3H), 2.00 (s, 3H), 1.95
(s, 3H), 0.04 (s, 6H, Me2Si). LC-HRESIMS m/z 1173.5968 [M+Na]+
(calcd for C59H94O20NaSi 1173.6005).
Compound 7: 1H NMR (ı, CDCl3, 300 MHz): 5.32 (t, 1H, J = 9.9 Hz,
H-3ꢀ), 5.25 (d, 1H, J = 6.0 Hz, H-6), 5.17 (dd, 1H, J = 3.3, 10.2 Hz, H-3ꢀꢀ),
5.06 (t, 1H, J = 9.9 Hz, H-4ꢀ), 5.04–5.06 (m, 1H, H-2ꢀꢀ), 4.98 (d, 1H,
J = 3.0 Hz, H-1ꢀ), 4.95 (t, 1H, J = 9.9 Hz, H-4ꢀꢀ), 4.91 (d, 1H, J = 1.2 Hz,
H = 1ꢀꢀ), 4.42–4.48 (m, 1H, H-16), 4.28 (dd, 1H, J = 3.9, 12.3 Hz, H-
6aꢀ), 4.01–4.05 (m, 1H, H-6bꢀ), 3.80–3.97 (m, 5H, H-26, 22, 2ꢀ, 5ꢀ),
2.2.4. 2,3,4-Tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-tri-
O-acetyl-˛-d-glucopyranosyl trichloroacetimidate (compound
5)
To a solution of carefully dried 4 (1.10 g, 1.77 mmol) in dry
CH2Cl2 (20 ml) under argon was added NIS (600 mg, 2.65 mmol)

20
H. Wang et al. / Steroids 76 (2011) 18–27
3.64–3.73 (m, 1H, H-5ꢀꢀ), 3.41–3.52 (m, 1H, H-3), 2.11 (s, 3H), 2.08 (s,
3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H),
0.04 (s, 6H, Me2Si). 13C NMR (ı, CDCl3, 150 MHz): 171.34, 170.64,
170.11, 169.98, 169.90, 169.71, 169.27, 141.81 (C-5), 120.77 (C-6),
98.91 (C-1ꢀ), 94.39 (C-1ꢀꢀ), 75.95 (C-2ꢀ), 75.14 (C-22), 73.46 (C-16),
72.44 (C-3), 71.79 (C-3ꢀ), 70.87 (C-4ꢀ), 69.95 (C-2ꢀꢀ), 69.21 (C-26),
68.84 (C-5ꢀꢀ), 68.52 (C-4ꢀꢀ), 68.39 (C-3ꢀꢀ), 67.19 (C-5ꢀ), 61.92 (C-6ꢀ),
56.47, 54.58, 50.09, 42.68, 42.17, 39.72, 37.26, 36.49, 35.04, 33.32,
33.20, 33.04, 31.98, 31.61, 31.57, 30.40, 25.90 (3C), 20.95, 20.81,
20.77, 20.71, 20.70, 20.57 (2C), 20.48, 19.14, 18.22, 17.19, 17.15,
12.85, 11.16, −4.61 (2C). LC-HRESIMS m/z 1151.6271 [M+H]+ (calcd
for C59H95O20Si 1151.6186).
(1 M) was added. The reaction was refluxed at 80 ◦C for 24 h, neu-
tralized to neutral with Amberlite® IR-120 (H+), and filtered. The
filtrate was concentrated in vacuo. The residue was purified with
sephadex LH-20 column chromatography (MeOH) to afford com-
pound 11 (15 mg, 88% two steps).
1H NMR (ı, CDCl3, 600 MHz): 6.40 (s, 1H, H-1ꢀꢀ), 5.32 (d, 1H,
J = 4.2 Hz, H-6), 4.77–4.81 (m, 1H, H-2ꢀꢀ), 4.73 (dd, 1H, J = 3.0, 9.6 Hz,
H-3ꢀꢀ), 4.13–4.48 (m, 8H, H-22, 1ꢀ, 2ꢀ, 3ꢀ, 4ꢀ, 6aꢀ, 6bꢀ, 4ꢀꢀ), 3.70–3.82 (m,
5H, H-3, 26, 5ꢀ, 5ꢀꢀ), 1.35 (d, 3H, J = 6.6 Hz, H-6ꢀꢀ), 1.10 (d, 3H, J = 6.6 Hz,
H-27), 1.02 (s, 3H), 0.80 (s, 3H). LC-HRESIMS m/z 763.4229 [M+Na]+
(calcd for C39H64O13Na 763.4244).
Compound 8: 1H NMR (ı, CDCl3, 300 MHz): 5.30 (d, 1H, J = 3.9 Hz,
H-6), 5.22 (t, 1H, J = 9.3 Hz, H-3ꢀ), 5.13–5.01 (m, 4H, H-1ꢀꢀ, 2ꢀꢀ, 3ꢀꢀ, 4ꢀꢀ),
4.94 (t, 1H, J = 9.3 Hz, H-4ꢀ), 4.48 (d, 1H, J = 7.8 Hz, H-1ꢀ), 4.16–4.30
(m, 2H, H-16, 5ꢀ), 3.72–4.09 (m, 5H, H-22, 26, 6aꢀ, 6bꢀ), 3.68–3.70
(m, 1H, H-2ꢀ), 3.55–3.62 (m, 1H, H-5ꢀꢀ), 3.43–3.48 (m, 1H, H-3), 2.12
(s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s,
3H), 1.97 (s, 3H), 0.04 (s, 6H, Me2Si). LC-HRESIMS m/z 1151.6274
[M+H]+ (calcd for C59H95O20Si 1151.6186).
2.2.8. (25R)-26-Acetyloxy-3ˇ-tert-butyldimethylsilyloxy-16ˇ-
hydroxycholest-5-ene-22-one
16-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-˛-d-glucopyranoside] (compound
12)
Following the same procedure as compound 9, compound 7
(60 mg, 0.051 mmol) was converted to compound 12 (50 mg, 83%).
1H NMR (ı, CDCl3, 600 MHz): 5.29 (d, 1H, J = 3.0 Hz, H-6), 5.22
(t, 1H, J = 9.6, H-3ꢀ), 5.21 (dd, 1H, J = 3.6, 10.2 Hz, H-3ꢀꢀ), 5.03 (t, 1H,
J = 9.6 Hz, H-4ꢀ), 4.99 (dd, 1H, J = 1.2 Hz, H-2ꢀꢀ), 4.93 (t, 1H, J = 10.2 Hz,
H-4ꢀꢀ), 4.91 (d, 1H, J = 3.6 Hz, H-1ꢀ), 4.83 (s, 1H, H-1ꢀꢀ), 4.39 (q, 1H,
J = 5.4, 13.2 Hz, H-16), 4.28 (dd, 1H, J = 3.6, 12.6 Hz, H-6aꢀ), 4.04
(d, 1H, J = 12.0 Hz, H-6bꢀ), 3.93 (m, 2H, H-26), 3.87 (m, 1H, H-5ꢀ),
3.74 (dd, 1H, J = 3.6, 9.6 Hz, H-2ꢀ), 3.55 (d, 1H, J = 9.6 Hz, H-5ꢀꢀ), 3.46
(m, 1H, H-3), 3.04–3.10 (m, 1H), 2.93–2.98 (m, 1H), 2.54–2.61 (m,
1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.99 (s,
3H), 1.98 (s, 3H), 1.95 (s, 3H), 0.04 (s, 6H, Me2Si). 13C NMR (ı,
CDCl3, 150 MHz): 213.65 (C-22), 171.18, 170.60, 170.31, 169.99,
169.79, 169.59, 169.51, 141.63 (C-5), 120.69 (C-6), 98.97 (C-1ꢀꢀ),
93.31 (C-1ꢀ), 75.84 (C-2ꢀ), 74.24 (C-16), 72.43 (C-3), 71.80 (C-3ꢀ),
71.07 (C-4ꢀ), 70.04 (C-2ꢀꢀ), 69.15 (C-26), 68.21 (C-3ꢀꢀ), 68.14 (C-
4ꢀꢀ), 67.92 (C-5ꢀꢀ), 66.96 (C-5ꢀ), 61.43 (C-6ꢀ), 54.98, 53.40, 49.86,
43.41, 42.70, 41.63, 39.88, 37.74, 37.22, 36.46, 31.99, 31.97, 31.84,
31.65, 31.60, 26.94, 25.90 (3C), 20.93, 20.84, 20.81, 20.72, 20.68,
20.62, 20.58, 20.50, 19.35, 18.23, 17.06, 17.08, 16.75, 13.50, −4.62
(2C). LC-HRESIMS m/z 1171.5868 [M+Na]+ (calcd for C59H92O20NaSi
1171.5843).
2.2.6. (22S,25R)-26-Acetyloxy-3ˇ-tert-butyldimethylsilyloxy-22-
hydroxycholest-5-ene-16-one
22-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-ˇ-d-glucopyranoside] (compound
9)
To a solution of compound 6 (20 mg, 0.017 mmol) in CH2Cl2
(2 ml) was added pyridinium dichromate (13 mg, 0.035 mmol).
The reaction was stirred at room temperature for 48 h and was
quenched with saturated NaSO3. The organic layer was separated
and the water layer was extracted with CH2Cl2 three times. The
combined organic layer was washed with saturated NaHCO3 and
brine, dried with anhydrous NaSO4, and then filtered. The filtrate
was concentrated in vacuo and purified by column chromatography
to yield compound 9 (15 mg, 67%).
1H NMR (ı, CDCl3, 300 MHz): 5.33 (d, 1H, J = 3.9 Hz, H-6), 5.22
(t, 1H, J = 9.3 Hz, H-3ꢀ), 5.15–5.19 (m, 1H, H-3ꢀꢀ), 5.03–5.09 (m,
3H, H-1ꢀꢀ,2ꢀꢀ,4ꢀꢀ), 4.94 (t, 1H, J = 9.3 Hz, H-4ꢀ), 4.45 (d, 1H, J = 7.8 Hz,
H-1ꢀ), 4.07–4.31 (m, 4H, H-22, 5ꢀ, 6aꢀ, 6bꢀ), 3.85–3.97 (m, 2H, H-
26), 3.66–3.71 (m, 1H, H-2ꢀ), 3.61–3.64 (m, 1H, H-5ꢀꢀ), 3.45–3.58
(m, 1H, H-3), 2.12 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s,
3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H), 0.06 (s, 6H, Me2Si).
13C NMR (ı, CDCl3, 150 MHz): 219.92 (C-16), 171.22, 170.64,
170.42, 169.98, 169.88, 169.71, 169.32, 141.45 (C-5), 120.64 (C-6),
101.72 (C-1ꢀ), 96.87 (C-1ꢀꢀ), 79.13 (C-2ꢀ), 74.98, 74.65, 72.37, 71.66,
70.95, 69.52, 69.31, 69.11, 68.66, 66.67, 62.83, 62.13, 49.88, 49.15,
43.01, 42.71, 39.19, 38.90, 36.97, 36.61, 34.66, 32.40, 31.97, 31.58,
31.02, 30.82, 29.23, 25.92 (3C), 20.97, 20.85, 20.77, 20.73, 20.70,
20.64, 20.61, 19.40, 18.24, 17.16, 16.65, 14.11, 12.89, 11.35, −4.58
(2C). LC-HRESIMS m/z 1149.6113 [M+H]+ (calcd for C59H93O20Si
1149.6024).
2.2.9.
(25R)-26-Acetyloxy-3ˇ,16ˇ-dihydroxycholest-5-ene-22-one
16-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
tri-O-acetyl-˛-d-glucopyranoside] (compound
13)
Following the same procedure as compound 10, compound 12
(53 mg, 0.046 mmol) was converted to compound 13 (45 mg, 92%).
1H NMR (ı, CDCl3, 600 MHz): 5.32 (d, 1H, J = 4.8 Hz, H-6), 5.21
(m, 2H, H-3ꢀ, 3ꢀꢀ), 5.02 (t, 1H, J = 10.2 Hz, H-4ꢀ), 4.98 (dd, 1H, J = 1.2,
3.0 Hz, H-2ꢀꢀ), 4.92 (t, 1H, J = 10.2 Hz, H-4ꢀꢀ), 4.90 (d, 1H, J = 3.6 Hz,
H-1ꢀ), 4.82 (s, 1H, H-1ꢀꢀ), 4.38 (q, 1H, J = 4.8, 12.6 Hz, H-16), 4.27 (dd,
1H, J = 3.2, 12.6 Hz, H-6aꢀ), 4.03 (d, J = 12.0 Hz, H-6bꢀ), 3.91–3.92 (m,
2H, H-26), 3.85–3.86 (m, 1H, H-5ꢀ), 3.73 (dd, 1H, J = 3.6, 10.8 Hz, H-
2ꢀ), 3.54 (d, 1H, J = 10.2 Hz, H-5ꢀꢀ), 3.48–3.53 (m, 1H, H-5), 3.04–3.09
(m, 1H), 2.92–2.98 (m, 1H), 2.54–2.59 (m, 1H), 2.27 (dd, 1H,
J = 3.6, 13.2 Hz), 2.11 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.04 (s,
3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H). 13C NMR (ı, CDCl3,
150 MHz): 213.64 (C-22), 171.18, 170.58, 170.29, 169.98, 169.75,
169.57, 169.50, 140.86 (C-5), 121.17 (C-6), 98.96 (C-1ꢀꢀ), 93.28 (C-
1ꢀ), 75.87, 74.18, 71.75, 71.52, 71.09, 70.02, 69.13, 68.18, 68.11,
67.90, 66.93, 61.40, 54.95, 53.32, 49.74, 43.37, 42.07, 41.59, 39.82,
37.72, 37.07, 36.36, 31.97, 31.79, 31.59, 31.57, 31.48, 26.91, 20.90,
20.81 (2C), 20.70, 20.66, 20.61, 20.56, 20.48, 19.30, 17.03 (2C), 16.72,
13.47. LC-HRESIMS m/z 1057.5116 [M+Na]+ (calcd for C53H78O20Na
1057.49841).
2.2.7. (22S,25R)-3ˇ,22,26-Trihydroxycholest-5-ene-16-one
22-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-ˇ-d-glucopyranoside]
(compound 11)
To
a solution of compound 9 (26 mg, 0.023 mmol) in
MeOH–CH2Cl2 (4:1, 2 ml) was added p-toluenesulfonic acid mono-
hydrate (0.4 mg, 0.0023 mmol). The reaction was stirred at room
temperature for 6 h and was quenched with saturated NaHCO3.
The organic layer was separated and the water layer was extracted
with CH2Cl2 three times. The combined organic layer was washed
with saturated NaHCO3 and brine, dried with anhydrous NaSO4,
and then filtered. The filtrate was concentrated in vacuo to give a
residue and purified by silica gel column chromatography to afford
compound 10 (22 mg, 94%), which was dissolved in MeOH–CH2Cl2
(1:1, 2 ml), and then catalytic amount of NaOMe/MeOH solution

H. Wang et al. / Steroids 76 (2011) 18–27
21
2.2.10. (25R)-3ˇ,16ˇ,26-Trihydroxy-cholest-5-ene-22-one
16-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-˛-d-glucopyranoside]
(compound 14)
Following the same procedure as compound 11 from 10, com-
pound 13 (45 mg, 0.043 mmol) was converted to compound 14
(30 mg, 94%).
monohydrate. The reaction was stirred at room temperature for 6 h
and was quenched with saturated NaHCO3. The organic layer was
separated and the water layer was extracted with CH2Cl2 three
times. The combined organic layer was washed with saturated
NaHCO3 and brine, dried with anhydrous NaSO4, and then filtered.
The filtrate was concentrated in vacuo to give a residue. The residue
was dissolved in pyridine (60 ml) at 0 ◦C (ice bath), benzoyl chloride
(1.66 ml, 14.32 mmol) was added. The reaction was stirred for 24 h
and concentrated in vacuo to give a residue. The residue was puri-
fied by column chromatography to yield compound 18 (5.12 g, 93%).
1H NMR (ı, CDCl3, 300 MHz): 8.04 (d, 2H, J = 7.6 Hz, Ph), 7.55 (t,
1H, J = 7.6 Hz, Ph), 7.43 (t, 2H, J = 7.6 Hz, Ph), 5.42 (d, 1H, J = 5.1 Hz,
H-6), 4.82–4.92 (m, 1H, H-3), 3.93–3.96 (m, 2H, H-26), 2.17 (s, 3H),
2.05 (s, 3H), 1.10 (s, 3H), 0.96 (d, 3H, J = 6.6 Hz), 0.81 (s, 3H). 13C
NMR (ı, CDCl3, 100 MHz): 218.02 (C-16), 213.33 (C-22), 171.37 (Bz),
165.93 (Ac), 139.81 (C-5), 133.63, 132.73, 130.11, 129.47, 128.40,
128.22, 121.88 (C-6), 74.26 (C-3), 69.01 (C-26), 66.10 (C-17), 51.09,
49.53, 43.31, 41.61, 39.63, 38.49, 38.06, 37.13, 36.67, 36.64, 32.00,
31.68, 30.87, 27.71, 26.64, 20.91, 20.45, 19.32, 16.76, 15.33, 12.93.
LC-HRESIMS m/z 577.3532 [M+H]+ (calcd for C36H49O6 577.3532).
m.p.: 173.0–175.0 ◦C. 1H NMR (ı, C5D5N, 600 MHz): 5.89 (s, 1H,
H-1ꢀꢀ), 5.39 (d, 1H, J = 4.8 Hz, H-6), 5.37 (d, 1H, J = 3.6 Hz, H-1ꢀ), 4.81
(m, 1H, H-2ꢀꢀ), 4.74 (q, 1H, J = 5.4, 12.6 Hz, H-16), 4.51 (dd, 1H, J = 3.6,
9.6 Hz, H-6aꢀ), 4.37–4.42 (m, 3H, H-5ꢀ, 6bꢀ, 3ꢀꢀ), 4.23–4.27 (m, 2H,
H-4ꢀ, 4ꢀꢀ), 4.13 (dd, 1H, J = 3.6, 9.6 Hz, H-2ꢀ), 3.90–3.93 (m, 1H, H-
5ꢀꢀ), 3.82–3.87 (m, 1H, H-3), 3.65–3.76 (m, 2H, H-26), 1.59 (d, 3H,
J = 6.0 Hz), 1.23 (s, 3H), 1.11 (d, 3H, J = 3.6 Hz), 1.07 (s, 3H). 13C NMR
(ı, C5D5N, 150 MHz): 213.39 (C-22), 141.88 (C-5), 121.37 (C-6),
104.40, 95.02, 79.74, 74.40, 74.14, 73.92, 73.87, 72.67, 72.22, 71.78,
71.30, 70.02, 67.36, 62.42, 55.85, 54.26, 50.54, 43.79, 43.62, 42.10,
40.46, 38.47, 37.84, 36.98, 36.71, 32.70, 32.16, 31.80, 30.01, 27.61,
21.26, 19.61, 18.37, 17.31, 17.21, 14.19. LC-HRESIMS m/z 763.4333
[M+Na]+ (calcd for C39H64O13Na 763.4244).
2.2.11. (25R)-26-Acetyloxy-3ˇ-tert-butyldimethylsilyloxy-16ˇ-
hydroxycholest-5-ene-22-one
2.2.14. (22R,25R)-26-Acetyloxy-3ˇ-benzoyloxyfurost-
16-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-(1 → 2)-3,4,6-
5-ene-22-ol (compound 19)
tri-O-acetyl-ˇ-d-glucopyranoside] (compound
To a solution of compound 18 (1.6 g, 2.77 mmol) in dry THF
(50 ml) under argon cooled to −15 ◦C, NaBH4 (1.16 g, 30.5 mmol)
and CeCl3·7H2O (1.45 g, 3.88 mmol) were added. The reaction mix-
ture was stirred for 16 h, then cooled to −40 ◦C and quenched
with methyl alcohol. The suspension was diluted with CH2Cl2.
The organic layer was washed with saturated NaHCO3 and brine
respectively, and then dried over anhydrous NaSO4, and filtered.
The filtrate was concentrated in vacuo to give a residue, which
was chromatographed on a silica gel column to afford furostan
sapogenin 19 (1.1 g, 69%, white solid).
15)
Following the same procedure as compound 9, compound 8
(13 mg, 0.0113 mmol) was converted to compound 15 (10 mg, 77%).
1H NMR (ı, CDCl3, 600 MHz): 5.38 (dd, 1H, J = 3.6, 9.6 Hz, H-3ꢀꢀ),
5.30 (d, 1H, J = 5.4 Hz, H-6), 5.14 (t, 1H, J = 9.6 Hz, H-3ꢀ), 5.09 (s, H-
1ꢀꢀ), 5.07 (t, 1H, J = 10.2 Hz, H-4ꢀ), 5.01 (m, 1H, H-2ꢀꢀ), 4.92 (t, 1H,
J = 9.6 Hz, H-4ꢀꢀ), 4.23 (dd, 1H, J = 5.4, 7.2 Hz, H-6aꢀ), 4.02–4.06 (m,
2H, H-1ꢀ, 6bꢀ), 3.92–3.93 (m, 2H, H-26), 3.85–3.89 (m, 1H, H-5ꢀ),
3.73–3.76 (m, 1H, H-2ꢀ), 3.62–3.64 (m, 1H, H-5ꢀꢀ), 3.45–3.49 (m, 1H,
H-3), 2.12 (s, 3H), 2.07 (s, 3H), 2.05 (s, 6H), 2.00 (s, 6H), 1.98 (s, 3H),
0.05 (s, 6H, Me2Si). 13C NMR (ı, CDCl3, 150 MHz): 214.68 (C-22),
171.22, 170.58, 169.93, 169.87, 169.71 (2C), 169.65, 141.46 (C-5),
120.84 (C-6), 101.55 (C-1ꢀ), 96.85 (C-1ꢀꢀ), 80.22 (C-2ꢀ), 74.94, 73.90,
72.61, 72.10, 71.55, 69.76, 69.41, 69.16, 68.33, 66.40, 62.19, 55.91,
54.34, 50.07, 43.95, 42.75, 41.61, 39.29, 38.40, 37.29, 36.51, 32.02,
31.87, 31.66, 31.31, 29.68, 26.77, 25.92 (3C), 20.97 (2C), 20.84 (2C),
20.75 (2C), 20.68 (2C), 19.48, 18.24, 17.65, 16.62, 16.15, 13.43, −4.60
(2C). LC-HRESIMS m/z 1149.6122 [M+H]+ (calcd for C59H93O20Si
1149.6024).
1H NMR (ı, C5D5N, 300 MHz): 8.25 (d, 2H, J = 7.2 Hz, Ph),
7.44–7.55 (m, 3H, Ph), 5.37 (s, 1H, H-6), 4.98–5.04 (m, 2H, H-3,
16), 3.95–4.12 (m, 2H, H-26), 1.96 (s, 3H), 1.34 (d, 3H, J = 6.6 Hz),
1.02 (s, 3H), 0.92 (s, 3H), 0.91 (d, 3H, J = 6.6 Hz). 13C NMR (ı, C5D5N,
100 MHz): 170.77 (Ac), 165.98 (Bz), 139.90 (C-5), 133.28, 131.39,
129.92 (2C), 128.86 (2C), 122.83 (C-6), 110.49 (C-22), 81.15 (C-16),
74.84 (C-3), 69.38 (C-26), 63.78 (C-17), 56.54 (C-14), 50.17 (C-9),
40.82, 40.70, 39.91, 38.52, 37.19, 37.07, 36.98, 33.31, 32.45, 32.28,
31.63, 28.22, 28.14, 21.12, 20.75, 19.38, 17.04, 16.49, 16.41. LC-
HRESIMS m/z 601.3582 [M+Na]+ (calcd for C36H50O6Na 601.3505).
2.2.12. (25R)-3ˇ,16ˇ,26-Trihydroxy-cholest-5-ene-22-one
16-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-ˇ-d-glucopyranoside]
(compound 17)
Following the same procedure as compound 11, compound 15
(26 mg, 0.023 mmol) was converted to compound 17 (16 mg, 94%
two steps).
2.2.15.
(22S,25R)-26-Acetyloxy-3ˇ-benzoyloxyfurost-5-ene-22-ol
22-O-(3,4,6-tetra-O-acetyl-˛-d-glucopyranoside) (compound 21)
and (22R,25R)-26-acetyloxy-3ˇ-benzoyloxyfurost-5-ene-22-ol
22-O-(2,3,4,6-tetra-O-acetyl-˛-d-glucopyranoside) (compound
22)
1H NMR (ı, C5D5N, 600 MHz): 6.64 (s, 1H, H-1ꢀꢀ), 5.33 (d, 1H,
J = 4.8 Hz, H-6), 4.84 (m, 1H, H-2ꢀꢀ), 4.76–4.78 (m, 2H, H-16, 3ꢀꢀ), 4.56
(d, 1H, J = 7.8 Hz, H-1ꢀ), 4.12–4.49 (m, 7H, H-2ꢀ, 3ꢀ, 4ꢀ, 5ꢀ, 6aꢀ, 6bꢀ, 4ꢀꢀ),
3.80–3,85 (m, 1H, H-3), 1.75 (d, 1H, J = 6.0 Hz), 1.18 (s, 3H), 1.08 (d,
3H, J = 6.6 Hz), 1.00 (s, 3H). 13C NMR (ı, C5D5N, 150 MHz): 214.95 (C-
22), 141.92 (C-5), 121.26 (C-6), 103.39, 101.93, 79.91, 19.70, 78.69,
77.06, 74.18, 72.73, 72.38, 72.27, 71.27, 69.62, 67.60, 62.72, 57.09,
54.86, 50.55, 43.93, 43.53, 42.09, 39.84, 39.69, 37.76, 37.64, 36.92,
36.39, 32.66, 32.08, 31.74, 27.85, 21.10, 19.66, 19.07, 17.40, 16.71,
14.03. LC-HRESIMS m/z 763.4309 [M+Na]+ (calcd for C39H64O13Na
763.4329).
A solution of carefully dried furostan sapogenin 19 (128 mg,
0.218 mmol) and carefully dried monosaccharide trichloroacetimi-
date 20 (107 mg, 0.218 mmol) in dry CH2Cl2 (6 ml) was stirred with
◦
˚
4 A molecular sieves (100 mg) under argon at −20 C for 15 min, and
then TMSOTf (3.9 ␮l, 0.0218 mmol) was added. The reaction mix-
ture was stirred for an additional 1 h, quenched with triethylamine,
diluted with CH2Cl2. The molecular sieves were filtered out. The fil-
trate was washed with saturated NaHCO3 and brine respectively,
then dried over anhydrous NaSO4, and filtered. The filtrate was con-
centrated in vacuo to give a residue, which was chromatographed
on a silica gel column to afford two products: compound 21 (55 mg,
28%), compound 22 (58 mg, 29%).
2.2.13. (25R)-26-acetyloxy-3ˇ-benzoyloxycholest-
5-ene-16,22-dione (compound 18)
To a solution of compound 1 (5.6 g, 9.55 mmol) in CH2Cl2–MeOH
(1:3, 80 ml) was added catalytic amount of p-toluenesulfonic acid
Compound 21: m.p.: 65.8–67.8 ◦C. 1H NMR (ı, C5D5N, 600 MHz):
8.24 (d, 2H, J = 7.2 Hz, Ph), 7.57 (t, 1H, J = 7.6 Hz, Ph), 7.46 (t, 2H,
J = 7.6 Hz, Ph), 6.04 (t, 1H, J = 9.6 Hz, H-3ꢀ), 5.87 (d, 1H, J = 3.6 Hz, H-
1ꢀ), 5.50 (t, 1H, J = 9.6 Hz, H-4ꢀ), 5.36–5.38 (m, 2H, H-6, 2ꢀ), 5.00–5.05

22
H. Wang et al. / Steroids 76 (2011) 18–27
(m, 1H, H-16), 4.91–4.99 (m, 1H, H-3), 4.61–4.64 (m, 1H, H-5ꢀ),
4.46–4.56 (m, 2H, H-6aꢀ, 6bꢀ), 4.01–4.13 (m, 2H, H-26), 2.15 (s, 3H,
Ac), 2.13 (s, 3H, Ac), 2.08 (s, 6H, Ac), 2.04 (s, 3H, Ac), 1.20 (d, 3H,
J = 6.6 Hz), 1.04 (s, 3H), 0.98 (d, 3H, J = 6.6 Hz), 0.85 (s, 3H). 13C NMR
(ı, C5D5N, 150 MHz): 170.82, 170.47 (2C), 170.41, 170.03, 166.01,
140.03 (C-5), 133.32, 131.38, 129.94 (2C), 128.89 (2C), 122.69 (C-6),
115.45 (C-22), 89.36 (C-1ꢀ), 83.05, 74.78, 71.58, 70.86, 69.89, 69.08,
68.71, 63.67, 62.29, 56.44, 50.21, 41.24, 39.80, 38.52, 37.16, 37.01,
32.26, 32.45, 32.33, 32.29, 31.43, 28.14 (2C), 21.07, 20.78 (3C), 20.61,
20.56, 20.55, 19.39, 16.84, 16.48, 15.40. LC-HRESIMS m/z 931.4524
[M+Na]+ (calcd for C50H68O15Na 931.4455).
˚
5 (130 mg, 0.18 mmol) in dry CH2Cl2 (6 ml) was stirred with 4 A
molecular sieves (100 mg) under argon at −20 ◦C for 15 min, and
then TMSOTf (3.3 ␮l, 0.018 mmol) was added. The reaction mix-
ture was stirred for 2 h, quenched with triethylamine, diluted with
CH2Cl2. The molecular sieves were filtered out. The filtrate was con-
centrated in vacuo to give a residue, which was chromatographed
on a silica gel column to afford compound 25 (64 mg, 32%).
1H NMR (ı, C5D5N, 300 MHz): 8.26 (d, 2H, J = 7.8 Hz, Ph), 7.54 (t,
1H, J = 7.6 Hz, Ph), 7.46 (t, 2H, J = 7.8 Hz, Ph), 6.12 (t, 1H, J = 9.6 Hz,
H-3ꢀ), 5.78 (d, 1H, J = 3.6 Hz, H-1ꢀ), 5.48–5.65 (m, 4H, H-4ꢀ, 2ꢀꢀ, 3ꢀꢀ,
4ꢀꢀ), 5.35 (m, 2H, H-6, 1ꢀꢀ), 4.87–5.13 (m, 2H, H-3, 16), 4.76 (dd,
1H, J = 4.8, 12.3 Hz, H-6aꢀ), 4.05–4.59 (m, 6H, H-26, 2ꢀ, 5ꢀ, 6bꢀ, 5ꢀꢀ),
2.21 (s, 3H, Ac), 2.19 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.05 (s, 6H, Ac),
2.04 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.39 (d, 3H, J = 6.0 Hz), 1.34 (d,
3H, J = 6.3 Hz), 1.06 (d, 3H, J = 6.9 Hz), 1.03 (s, 3H), 0.86 (s, 3H). 13C
NMR (ı, C5D5N, 150 MHz): 170.91, 170.61, 170.32, 170.18, 170.04,
169.85, 166.03, 164.54, 140.15 (C-5), 133.33, 131.39, 129.95 (2C),
128.90 (2C), 123.16 (C-6), 119.68 (C-22), 99.18, 92.20, 90.51, 75.40,
74.87, 72.77, 72.57, 71.42, 70.47, 70.00, 69.30, 69.18, 68.54, 67.64,
63.01, 56.70, 49.88, 40.60, 38.81, 38.56, 37.20, 36.96, 36.67, 36.01,
33.57, 32.30, 31.35, 31.18, 29.22, 28.17, 20.82 (3C), 20.78 (2C),
20.59, 20.51 (2C), 20.44, 19.38, 17.55, 17.34, 15.34. LC-HRESIMS
m/z 1161.4956 [M+Na]+ (calcd for C60H82O21Na 1161.52408).
Compound 22: m.p.: 47.6–49.6 ◦C. 1H NMR (ı, C5D5N, 600 MHz):
8.24 (d, 2H, J = 7.2 Hz, Ph), 7.54 (t, 1H, J = 7.6 Hz, Ph), 7.46 (t, 2H,
J = 7.6 Hz, Ph), 6.85 (d, 1H, J = 3.6 Hz, H-1ꢀ), 5.98 (t, 1H, J = 10.2 Hz,
H-3ꢀ), 5.61 (t, 1H, J = 10.2 Hz, H-4ꢀ), 5.36 (d, 1H, J = 4.8 Hz, H-6),
4.99–5.05 (m, 1H, H-3), 4.73 (q, 1H, J = 7.8, 15.0 Hz, H-16), 4.28–4.54
(m, 3H, H-2ꢀ, 6aꢀ, 6bꢀ), 4.00–4.13 (m, 2H, H-26), 2.25 (s, 3H, Ac),
2.20 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.07 (s, 3H, Ac), 1.96 (s, 3H, Ac),
1.15 (d, 3H, J = 6.6 Hz), 1.00 (s, 3H), 0.95 (d, 3H, J = 6.6 Hz), 0.80 (s,
3H). 13C NMR (ı, C5D5N, 150 MHz): 170.85, 170.49, 170.08, 169.88,
169.77, 166.01, 139.89 (C-5), 133.31, 131.40, 129.95 (2C), 128.89
(2C), 122.76 (C-6), 114.82 (C-22), 91.58 (C-1ꢀ), 82.71, 74.85, 71.42,
69.85, 69.35, 69.01, 69.03, 63.07, 62.30, 56.20, 50.01, 41.07, 40.55,
39.50, 38.50, 37.16, 36.96, 33.13, 32.30, 32.15, 31.42, 31.32, 28.14,
27.86, 20.99, 20.91, 20.85 (2C), 20.79, 20.56, 19.35, 16.83, 16.10,
14.26. LC-HRESIMS m/z 931.4432 [M+Na]+ (calcd for C50H68O15Na
931.4450).
2.2.19. (22S,25R)-Furost-5-ene-3ˇ,22,26-triol
22-O-[O-˛-l-rhamnopyranosyl-(1 → 2)-˛-d-glucopyranoside]
(compound 26)
Following the same procedure as compound 23, compound 25
(32 mg, 0.028 mmol) was converted to compound 26 (18 mg, 86%).
1H NMR (ı, C5D5N, 600 MHz): 6.27 (s, 1H, H-1ꢀꢀ), 5.97 (d,
J = 3.0 Hz, 1H, H-1ꢀ), 5.29 (d, J = 4.2 Hz, 1H, H-6), 4.77 (m, 1H, H-2ꢀꢀ),
4.17–4.56 (m, 10H, H-16, 2ꢀ, 3ꢀ, 4ꢀ, 5ꢀ, 6aꢀ, 6bꢀ, 3ꢀꢀ, 4ꢀꢀ, 5ꢀꢀ), 3.78–3.93
(m, 3H, H-3, 26), 1.17 (s, 3H, H-6ꢀꢀ), 0.99 (s, 3H, H-19), 0.94 (d,
3H, J = 7.2 Hz, H-19), 0.92 (d, 3H, J = 7.2 Hz, H-27), 0.85 (s, 3H, H-
18). 13C NMR (ı, C5D5N, 150 MHz): 141.66 (C-5), 121.34 (C-6),
118.89 (C-22), 103.25 (C-1ꢀꢀ), 93.19 (C-1ꢀ), 90.37 (C-16), 76.96, 75.28,
74.49, 74.08, 72.90, 72.67, 72.59, 72.21, 71.32, 69.75, 67.34, 62.87,
56.87, 50.23, 43.60, 40.35, 39.31, 37.77, 37.40, 37.21, 36.96, 32.64,
32.23, 31.90, 31.64, 29.79, 20.95, 20.52, 19.58, 18.65, 17.35, 15.40,
12.24. LC-HRESIMS m/z 763.4235 [M+Na]+ (calcd for C39H64O13Na
763.4239).
2.2.16. (22S,25R)-Furost-5-ene-3ˇ,22,26-triol
22-O-(˛-d-glucopyranoside) (compound 23)
To
a solution of compound 21 (39 mg, 0.043 mmol) in
MeOH–CH2Cl2 (1:1, 3 ml) was added 0.26 ml NaOMe/MeOH solu-
tion (1 M). The reaction was refluxed at 80 ◦C for 48 h, and then
concentrated in vacuo. The residue was purified with sephadex LH-
20 column chromatography (MeOH) to afford compound 23 (23 mg,
92%).
1H NMR (ı, C5D5N, 600 MHz): 5.85 (d, 1H, J = 3.6 Hz, H-1ꢀ), 5.37
(q, 1H, J = 7.8, 15.0 Hz, H-16), 5.30 (d, 1H, J = 4.8 Hz, H-6), 4.55–4.62
(m, 2H), 4.37–4.41 (m, 1H), 4.11–4.16 (m, 2H), 3.71–3.83 (m, 4H, H-
3, 26, 5ꢀ). 13C NMR (ı, C5D5N, 150 MHz): 141.95 (C-5), 121.08 (C-6),
114.54 (C-22), 93.08 (C-1ꢀ), 82.55 (C-16), 75.56, 75.00, 73.71, 72.62,
71.27, 67.59, 63.33, 56.34, 50.36, 43.53, 41.13, 40.92, 39.80, 37.83,
37.18, 37.04, 34.06, 32.65, 32.43, 32.28, 31.63, 30.02, 28.14, 21.17,
19.60, 17.22, 16.49, 16.17. LC-HRESIMS m/z 617.3701 [M+Na]+
(calcd for C33H54O9Na 617.3665).
2.3. Conformational analysis
2.3.1. Conformational analysis of compounds 21 and 22
Molecular structures of the 22S isomer of 21 and 22R isomer of
22 were built using Sybyl 7.3 version Molecular Modeling Software.
Structural energy minimization was performed using the stan-
dard Tripos molecular mechanics force field and Gasteiger–Hückel
charge in the software, with a 0.001 kcal/mol energy gradient con-
vergence criterion on the Silicon Graphics IRIS Octane computer
system. Structure manipulations and analysis were carried out with
the set of tools provided with the SYBYL package. Molecular dynam-
ics simulations and the minimizations of structures over Cartesian
coordinates employed the Tripos force field.
2.2.17. (22R,25R)-Furost-5-ene-3ˇ,22,26-triol
22-O-(˛-d-glucopyranoside) (compound 24)
Following the same procedure as compound 23, compound 22
(42 mg, 0.046 mmol) was converted to compound 24 (16 mg, 93%).
1H NMR (ı, C5D5N, 600 MHz): 6.06 (d, 1H, J = 3.6 Hz, H-1ꢀ), 5.38
(q, 1H, J = 7.8, 15.0 Hz, H-16), 5.36 (d, 1H, J = 4.8 Hz, H-6), 4.12–4.78
(m, 5H, H-2ꢀ, 3ꢀ, 4ꢀ, 6aꢀ, 6bꢀ), 3.70–3.86 (m, 4H, H-3, 26, 5ꢀ). 13C
NMR (ı, C5D5N, 150 MHz): 142.01 (C-5), 121.05 (C-6), 114.80 (C-
22), 110.80 (C-1ꢀ), 81.93 (C-16), 75.56, 75.01, 73.72, 72.63, 71.28,
67.55, 63.57, 56.34, 50.48, 43.55, 40.87, 40.52, 39.87, 37.83, 37.61,
37.06, 34.07, 32.66, 32.35, 32.24, 31.78, 29.29, 28.46, 21.23, 19.62,
17.21, 16.49, 16.36. LC-HRESIMS m/z 617.3654 [M+Na]+ (calcd for
C33H54O9Na 617.3660).
2.3.2. Details of the simulation procedure
First a very fast and flexible systematic search was introduced,
which could guarantee a completion of the task: to enumerate all of
the different torsional rotational settings. Molecular structures of
the 22S isomer of 21 and 22R isomer of 22 were searched system-
atically by changing the value of two specified torsions between
oxygen atom at C-16 and anomeric proton, each conformers were
minimized the strain energy and the results were analyzed using
the Molecular Spreadsheet. The conformer of the minimal energy
2.2.18. (22R,25R)-26-Acetyloxy-3ˇ-benzoyloxyfurost-
5-ene-22-ol 22-O-[O-2,3,4-tri-O-acetyl-˛-l-rhamnopyranosyl-
(1 → 2)-3,4,6-tri-O-acetyl-ˇ-d-glucopyranoside] (compound
25)
A solution of carefully dried furostan sapogenin 19 (208 mg,
0.36 mmol) and carefully dried disaccharide trichloroacetimidate

H. Wang et al. / Steroids 76 (2011) 18–27
23
which obtained by systematically exploring different sets of start-
ing coordinates was regard as the global minimum. Molecular
dynamics (MD) simulation started from this conformer to main-
tain constant temperature (1000 K) during the simulation. A single
molecular dynamics trajectory at constant temperature was gen-
erated. The molecular dynamics trajectory was updated using a
modified Beeman method to integrate the Newtonian equations
of motion. The time interval for the dynamics steps was set to 1 fs,
and the total length of the simulated trajectory reached 1 ns. From
the generated trajectory, 500 structures, equally spaced in time,
were collected, with the interval between snapshots set to 2 ps.
Each structure from the generated ensemble was then subjected
to molecular dynamics simulated annealing computation. During
a 2 ps simulation, the temperature changed between the starting
Fig. 1. Chemical shift differences between 3a (S)-MTPA ester and 3b (R)-MTPA ester
ꢀı (Hz) = ı(S)-MTPA − ı(R)-MTPA
.
Scheme 1. The synthesis of cholestane and furostan sapogenins.
Scheme 2. (a) NIS, AgOTf, CH2Cl2, −15 ◦C → r.t. 1 h, 88% and (b) CCl3CN, DBU, CH2Cl2, 0 ◦C, 6 h, 80%.
˚
Scheme 3. (a) TMSOTf, 4 A MS, dry CH2Cl2, r.t.; (b) PDC, CH2Cl2, r.t.; (c). p-TsOH·H2O, MeOH/CH2Cl2, r.t. and (d) MeONa, MeOH/CH2Cl2, r.t.

24
H. Wang et al. / Steroids 76 (2011) 18–27
Scheme 4. (a) p-TsOH·H2O, MeOH/CH2Cl2, r.t., 6 h, then benzoyl chloride, pyridine, 0 ◦C, 24h, 93% two steps; (b) NaBH4/CeCl3·7H2O, dry THF, −15 ◦C, 16 h, 69%; (c) TMSOTf,
4 A MS, dry CH2Cl2, −20 ◦C, 1 h and (d) MeONa, MeOH/CH2Cl2, 80 ◦C, 48 h.
˚
value of 1000 K and the final value of 100 K. The linear scaling pro-
tocol was used. Finally, all structures were minimized in Cartesian
coordinate space with the value of the rms termination criterion
3. Results and discussion
At the beginning, intermediate (25R)-26-acetyloxy-3ˇ-tert-
butyldimethylsilyloxycholest-5-ene-16,22-dione 1 was reduced by
NaBH4/CeCl3·7H2O in THF under controlled conditions (Scheme 1).
In previous reports [11], it had been well documented that 16-
ketone was more reactive than 22-ketone, which predicated the
˚
set to 0.01 kcal/mol/A. The value of the dielectric constant in all
calculations was 1.0; the default value for the Tripos force field.
The final set of 500 minimized structures was then further ana-
lyzed.
Fig. 2. Comparison of distances between oxygen atom at C-16 and anomeric proton in conformers of two epimers (21 and 22): (A) epimer 21 and (B) epimer 22.

H. Wang et al. / Steroids 76 (2011) 18–27
25
reduction of 16-ketone was easier than 22-ketone. Eventually, the
reduction of compound 1 afforded two products: (22R,25R)-26-
acetyloxy-3ˇ-tert-butyldimethylsilyloxyfurost-5-ene-22-ol 2 and
(22S,25R)-26-acetyloxy-3ˇ-tert-butyldimethylsilyloxycholest-
The 16ˇ-O-glycosides (7 and 8) and 22-O-glycoside (6) were
readily distinguished by analysis of their 1H NMR spectra. The most
characteristic 1H signal of the 16ˇ-O-glycoside 7 was a doublet at
4.98 ppm (d, J = 3.0 Hz) corresponding to the anomeric proton. For
compound 8, anomeric proton signal shifted upfield to 4.48 ppm
(d, J = 7.8 Hz). For 22-O-glycoside 6, anomeric proton appeared at
4.66 ppm (d, J = 7.8 Hz).
All of the glycosidic products (6, 7and 8) were subjected to
the oxidation with pyridinium dichromate (PDC). The generated
ketones showed different chemical shifts in 13C NMR spectra. The
ketone carbon C-22 appeared at 213.65 ppm for compound 12 and
214.68 ppm for compound 15, while compound 9 showed a char-
acteristic 13C signal of ketone carbon C-16 at 219.92 ppm.
Desilylation with p-toluenesulfonic acid monohydrate (p-
TsOH·H2O) afforded 3-t-butyldimethylsilyl (TBDMS) deprotection
compounds 10, 13 and 16. A final step of the synthesis was the
deprotection of acetyl groups, which was achieved by treatment
with MeONa in MeOH/CH2Cl2. All novel cholestane saponin ana-
logues (11, 14 and 17) were obtained and fully characterized by
spectral analysis (1H, 13C NMR and HRMS).
5-ene-16ˇ,22-diol 3. The relatively steady compound
3 was
glycosylated firstly.
Before the glycosylation occurred, the absolute configuration of
C-22 hydroxy in compound 3 was ascertained above all. 16ˇ,22-
Dihydroxy compound 3 was converted to 22-(S)- and 22-(R)-˛-
methoxy-˛-(trifluoromethyl)phenylacetyl (MTPA) derivatives (3a
and 3b) following the application of classical Mosher’s method [12],
which allowed us to assign the absolute configuration of C-22 as S
(Fig. 1). The steric hindrance around the hydroxyl group of C-16 was
considered to prevent bulky MTPA group from being introduced in.
Thioglycoside [˛-l-Rhap-(1-2)-ˇ-d-Glcp] 4, which was prepared
in accordance with the procedure described in previous literature
[10], was transformed into trichloroacetimidate 5 in Scheme 2.
Treatment of the hydrolysate of thioglycoside 4 with trichloroace-
tonitrile and 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) afforded
disaccharide trichloroacetimidate 5 finally.
With compound 3 and disaccharide trichloroacetimidate 5
in hand, glycosylation in the presence of trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) was carried out (Scheme 3) [13].
Apart from a pair of epimeric 16ˇ-O-glycosides 7 and 8, 22-O-
glycoside 6 was formed also. During the glycosylation, disaccharide
donor may be attacked by either the oxygen atom at C-16 (for 16ˇ-
O-glycoside) or that at C-22 (for 22-O-glycoside). The proportion of
three products (6:7:8 = 3:14:3) was determined by the relative rate
of the competing reactions.
Subsequently, glycosylation of furostan was executed in
Scheme 4. In previous research [10,14], 22-hemiketal or ketal
in furostan was found to be very unstable in acid con-
dition. To avoid this side reaction of furostan glycosyla-
tion products in final deprotection of 3ˇ-TBDMS group with
acid reagents, 3ˇ-TBDMS group of (25R)-26-acetyloxy-3ˇ-tert-
butyldimethylsilyloxycholest-5-ene-16,22-dione
1 was substi-
tuted with benzoyl group firstly. Removal of 3ˇ-TBDMS group
with p-TsOH·H2O, succeeding protection of exposed 3ˇ-hydroxy
Fig. 3. The conformers of two epimers (21 and 22): (A) the biggest distance conformer of epimer 21 and (B) the smallest distance conformer of epimer 22.

26
H. Wang et al. / Steroids 76 (2011) 18–27
Scheme 5. The formation of two configurations at C-22.
Scheme 6. (a) TMSOTf, 4 ◦C MS, dry CH2Cl2, −20 ◦C, 2 h, 32% and (b) MeONa, MeOH/CH2Cl2, 80 ◦C, 48 h, 86%.
with benzoyl chloride, afforded compound 18. Reduction with
NaBH4/CeCl3·7H2O in THF, compound 19 was obtained in a sat-
isfactory yield in the end. Then compound 19 was glycosylated
with d-glucose trichloroacetimidate 20 in the presence of TMSOTf
[13], the total conversion was about 60% and a pair of epimeric
22-O-glycosides 21 and 22 was gained in almost equal amounts.
The glycosidic products were distinguished by NMR spectra. The
obvious evidence was the carbon signal of C-22 (it appeared
at 110.49 ppm in 19) in 13C NMR spectra, which appeared at
115.45 ppm in compound 21 and 114.82 ppm in compound 22.
Another characteristic was doublet of anomeric proton in 1H NMR
spectra, which appeared at 5.87 ppm (J = 3.6 Hz) in compound 21
and 6.85 ppm (J = 3.6 Hz) in compound 22 (Scheme 4).
The anomeric proton signal of compound 22 shifted to a very low
field at 6.85 ppm. To explain this phenomenon, exhaustive confor-
mational analysis [13] for two epimers (21 and 22) was carried out.
As some of the conformers were very similar to each other, to sim-
plify analysis, the values of dihedral angles between oxygen atom at
C-16 and anomeric proton measured in generated structures were
chosen as a criterion describing their similarity. Two conformers
were assumed identical if the largest difference observed between
the values of all of their matching dihedral angles was less than
2◦ and only one of them was further considered. Then after apply-
ing a cutoff of 5 kcal/mol, 19 conformers of compound 21 and 27
conformers of compound 22 were obtained at last (Fig. 2).
Inspection of Figs. 2 and 3 indicates that there was a distinctive
difference in conformational freedom between the two epimers.
The figure of compound 21 showed that quite substantial propor-
˚
tions of generated conformers had the distance of about ∼3.5 A
which is similar to the conformer of compound 21 in Fig. 3. While
the figure of compound 22 showed that quite substantial propor-
˚
tions of generated conformers had the distance of about ∼2.5 A,
which is similar to the conformer of compound 22 in Fig. 3. These
figures suggested that the distance between oxygen atom at C-16
and anomeric proton of compound 22 was quite closer than that of
compound 21. The electrophilic deshielding effect of oxygen atom
at C-16 induced the remarkable downfield shift of anomeric proton
in compound 22.
The sapogenins of compounds 21 and 22 displayed two config-
urations (22S and 22R) at C-22. The epimerization of C-22 can be
explained as the same process of the mutarotation of aldoses and
ketoses [15,16] following acid catalysis (Scheme 5). The mutarota-
tion of C-22 occurred in the glycosylation of sapogenin 19 catalyzed
by Lewis acid TMSOTf.
Table 1
The in vitro cytotoxicities (IC50, ␮M) of synthetic cholestane and furostan saponin analogues.a
Cell lines
Dioscin
Cholestane saponin
Cholestane saponin
Cholestane saponin
Furostan saponin
Furostan saponin
Furostan saponin
analogues 11
analogues 14
analogues 17
analogues 23
analogues 24
analogues 26
BxPC3
U251
HEPG2
MCF-7
5.53
2.79
4.15
3.35
>100
>100
>100
>100
>100
15.8
16.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
50.3
47.7
45.9
>100
>100
>100
>100
>100
a
The in vitro cytotoxic activities against BxPC3 (pancreatic cancer), U251 (glioblastoma), HEPG2 (hepatocellular cancer) and MCF-7 (breast cancer) cell lines were evaluated
by the standard MTT assay using dioscin, a natural product, as a positive control.

H. Wang et al. / Steroids 76 (2011) 18–27
27
The epimers were respectively subjected to deprotection of
acetyl groups and benzoyl group with MeONa to afford furostan
saponin analogues 23 and 24 ultimately.
[2] Simões CMO, Amoros M, Girre L. Mechanism of antiviral activity of triterpenoid
saponins. Phytother Res 1999;13:323–8.
[3] Killeen GF, Madigan CA, Connolly CR, Walsh GA, Clark C, Hynes MJ, et
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in vitro properties for their in vivo impact. J Agric Food Chem 1998;46:
3178–86.
[4] Sindambiwe JB, Calomme M, Geerts S, Pieters L, Vlietinck AJ, Vanden Berghe DA.
Evaluation of biological activities of triterpenoid saponins from Maesa lanceo-
lata. J Nat Prod 1998;61:585–90.
[5] Li DW, Lee EB, Kang SS, Hyun JE, Whang WK. Activity-guided isolation of
saponins from Kalopanax pictus with anti-inflammatory activity. Chem Pharm
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[6] Itabashi M, Segawa K, Ikeda Y, Kondo S, Naganawa H, Koyano T, et al. A new
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istry 1992;31:3969–73.
[8] Hernádez JC, León F, Quintana J, Estévez F, Bermejo J. Icogenin, a new cyto-
toxic steroidal saponin isolated from Dracaena draco. Bioorg Med Chem
2004;12:4423–9.
Subsequently, glycosylation of compound 19 and disaccharide
trichloroacetimidate 5 was executed (Scheme 6) [13], and the 22-O-
glycoside 25 was gained eventually. The structure of glycosylation
product 25 was validated by NMR spectra. In 1H NMR spectra, char-
acteristic was doublet of anomeric proton at 5.78 ppm (J = 3.6 Hz).
In 13C NMR spectra, the carbon signal of C-22 was deshielded
to 119.68 ppm under the influence of glycosidic bond (in 19 it
appeared at 110.49 ppm). The protecting groups were removed in
the same way as described above (23 and 24) and a novel furostan
saponin analogue 26 was obtained.
The in vitro cytotoxicities of the synthetic cholestane and
furostan saponin analogues (11, 14, 17, 23, 24, 26) against BxPC3
(pancreatic cancer cells), U251 (glioblastoma cells), HEPG2 (hep-
atocellular cancer cells) and MCF-7 (breast cancer cells) were
evaluated by the standard MTT assay using dioscin as a positive
control. The results are listed in Table 1. Among the tested com-
pounds, only compounds 14 and 24 showed weak cytotoxicities to
U251 and HEPG2 cell lines, and compound 24 showed similar cyto-
toxicities to BxPC3 cell line at concentrations of 1–100 ␮M, which
were about 10–50 times less active than dioscin.
[9] Cheng MS, Wang QL, Tian Q, Song HY, Liu YX, Li Q, et al.
2003;68:3658–62.
J Org Chem
[10] Wang HX, Su FQ, Zhou L, Chen XG, Lei PS. Synthesis and cytotoxicities
of icogenin analogues with disaccharide residues. Bioorg Med Chem Lett
2009;19:2796–800.
[11] Kaufmann S, Rosenkranz G. Steroidal sapogenins. I. Transformation of kryp-
togenin into diosgenin and pseudodiosgenin.
3502–4.
J Am Chem Soc 1948;70:
[12] Takaashi Y, Mimaki Y, Kameyama A, Kuroda M, Sashida Y, Nikaido T, et al. Three
new cholestane bisdesmosides from Nolina recurvata stems and their inhibitory
activity on cAMP phosphodiesterase and Na+/K+ ATPase. Chem Pharm Bull
1995;43(7):1180–5.
Acknowledgements
[13] Wojtkielewicz A, Długosz M, Maj J, Morzycki JW, Nowakowski M, Renkiewicz
J, et al. New analogues of the potent cytotoxic saponin OSW-1. J Med Chem
2007;50:3667–73.
[14] Lee JS, Fuchs PL. Efficient protocol for ring opening of spiroketals
using trifluoroacetyl trifluoromethanesulfonate (TFAT). Org Lett 2003;5(20):
3619–22.
[15] Capon B. Mechanism in carbohydrate chemistry. Chem Rev 1969;69(4):
407–98.
[16] Silva AM, da Silva EC, da Silva CO. A theoretical study of glucose mutarotation
in aqueous solution. Carbohydr Res 2006;341:1029–40.
This work was supported by the National Natural Science
Foundation of China (30772632) and the National S&T Major
Special Project on Major New Drug Innovation (Item Number:
2009ZX09301-003).
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