C21 steroidal glycosides from Cynanchum wilfordii

Article abstract of DOI:10.1002/hlca.200900057

Eight new C₂₁ steroidal glycosides, named wilfosides A-H (1 - 8, resp.), along with one known compound wilfoside KIN (9), were isolated from the roots of Cynanchum wilfordii. The structures of the new glycosides were determined on the basis of

Full text of DOI:10.1002/hlca.200900057

Helvetica Chimica Acta – Vol. 92 (2009)
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C21 Steroidal Glycosides from Cynanchum wilfordii
by Wen-Juan Xianga), Lei Ma*a), and Li-Hong Hu*a)b)
a) School of Pharmacy, East China University of Science and Technology, 130 Meilong Road,
Shanghai 200237, P. R. China (phone: þ 86-21-50272221; fax: þ 86-21-50272221;
e-mail: malei@ecust.edu.cn; simmhulh@mail.shcnc.ac.cn)
b) Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of
Materia Medica, Shanghai 201203, P. R. China
Eight new C21 steroidal glycosides, named wilfosides A – H (1 – 8, resp.), along with one known
compound wilfoside KIN (9), were isolated from the roots of Cynanchum wilfordii. The structures of the
new glycosides were determined on the basis of spectroscopic analysis, including 1D- and 2D-NMR, and
ESI-MS techniques, as well as by comparison of the spectral data with those of related compounds.
Introduction. – Steroidal glycosides are widely distributed in the Cynanchum
species. Medicinal plants containing steroidal glycosides are commonly used for
medicinal purposes in many Asian countries. Modern biological studies have shown
that the extracts and fractions of Cynanchum species have varieties of biological
activities, including antitumor, antiepileptic, immunoregulation, anti-oxidation, and
antiviral effects [1]. Cynanchum wilfordii (Maxim.) Hemsl. (former Asclepiadaceae,
now Apocynaceae, subfamily Asclepiadoideae) is widely distributed in China and used
as traditional Chinese medicine for the treatment of impotency, neurasthenia, lumbago,
and abscesses [2]. C21 Steroidal glycosides have established themselves as an important
class of biologically active compounds in the Cynanchum species. Previous inves-
tigation on the C21 steroidal glycosides had shown the presence of normal and aberrant
pregnane skeletons [3 – 6]. The C21 steroidal glycosides with a normal pregnane
skeleton are sarcostin derivatives with an oligosaccharide at C(3) and acetyl, ikemaoyl,
cinnamoyl, isovaleroyl, p-hydrobenzoyl, and nicotinoyl esters at C(12) or C(20) [7].
Two sorts of aberrant pregnane glycosides with aglycone skeletons of the 13,14;14,15-
disecopregnane type and the 14,15-secopregnane type are known from the Cynanchum
species [8][9]. In these steroidal glycosides, the linkage sites of the sugar moieties are at
C(3) of their aglycones and the sugar portion is generally composed of a linear rather
than a branched oligosaccharide chain. The sugar residues include 2,6-dideoxysugar
units, 6-deoxysugar units, and glucose units with the mode of 1 ! 4 linkage [3 – 6].
As part of our phytochemical investigation of traditional Chinese medicinal plants,
we describe here the isolation and structure elucidation of eight new C21 steroidal
glycosides, wilfosides A – H (1 – 8, resp.), along with the known compound wilfoside
KIN (9) from the roots of C. wilfordii. Like many wilfosides [10], wilfosides A – F (1 – 6,
resp.) are sarcostin derivatives with an oligosaccharide at C(3) and esterified at C(20).
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Wilfosides G and H (7 and 8, resp.) have an 8,14-secopregnane skeleton previously
observed in Cynanchum species [8][9] and evidently arising from oxidative cleavage of
the 8,14 glycol of sarcotins. All compounds possessed an oligosaccharide chain
consisting of two, three, four, or five 2,6-dideoxysugar units at C(3) of the aglycone
[7][11]. The structures of the new glycosides were determined on the basis of
spectroscopic analysis, including 1D- and 2D-NMR and HR-ESI-MS techniques
(Fig. 1).
Results and Discussion. – Compounds 1 – 9 all showed positive Liebermann –
Burchard reactions, suggesting that they were steroidal glycosides, and positive
Keller– Killiani reactions, suggesting the presence of 2-deoxysugar moieties. Com-
1
pound 9 was elucidated to be wilfoside KIN by comparison of H- and 13C-NMR data
with the literature [12].
The identity of the monosaccharides in the hydrolysates after acid hydrolysis of
each compound was confirmed by co-TLC comparison with authentic sugars. For the
oleandrose and digitoxose, to the best of our knowledge, b-linked digitoxopyranosyl
and oleandropyranosyl units were so far only found in d-configuration in the
Asclepiadaceae family [10][13]. Due to their similar 1H- and 13C-NMR chemical shifts
with those reported in the literatures [9][10][14 – 16], all digitoxopyranosyl and
oleandropyranosyl units in this study were determined to be of d-configuration.
Wilfoside A (1) was isolated as a colorless, amorphous powder. HR-ESI-MS
indicated a molecular formula of C50H67NO14. The 13C-NMR and DEPT spectra
(Tables 1 and 2) showed signals of two CO groups, two C¼C bonds, and seven Me, nine
CH2, and 21 CH groups, as well as of seven quaternary C-atoms. The 1H-NMR of the
aglycone portion (Table 3) showed signals for three Me groups at d(H) 1.09 (s,
Me(19)), 1.34 (d, J ¼ 6.2, Me(21)), and 1.60 (s, Me(18)), and three signals at 3.52 – 3.55
(m, HꢀC(3)), 4.79 – 4.81 (m, HꢀC(12)), 4.85 – 4.87 (m, HꢀC(20)) corresponding to
secondary O-bearing C-atoms. The data were in good agreement with previously
published data for similar compounds [3][17][18], which indicated that the structure of
1 was based on the skeleton of sarcostin. The extensive 1D- and 2D-NMR study
revealed that the aglycone contained each a cinnamoyl and nicotinoyl moiety.
Diagnostic long-range correlations (HMBC) were observed between HꢀC(12) (d(H)
4.79 – 4.81) and C(1’cin) (d(C) 166.2), and between HꢀC(20) (d(H) 4.85 – 4.87) and
C(1’nic) (d(C) 163.7), revealing the connectivity between the cinnamoyl group and
C(12), and between the nicotinoyl group and C(20). The aglycone of 1 was determined
to be gagaminin by comparing with the NMR data of known C21 steroidal aglycones
[19]. The presence of gagaminin in the acid hydrolysate of 1 was confirmed by TLC
comparsion with an authentic sample.
The anomeric-C-atom resonances at d(C) 96.0 and 101.4 correlating with the
corresponding H-atoms at d(H) 4.83 (d, J ¼ 8.1) and 4.47 (d, J ¼ 7.8) in the HSQC
spectrum revealed the presence of two sugar residues. The coupling constants of the
anomeric H-atom signals indicated that 1 had two sugar units with b-linkages. The 1H-
and 13C-NMR spectra (Tables 4 and 2, resp.), and the TLC behavior of the two sugar
units suggested that the sugar moieties were b-cymaropyranosyl and b-oleandropyr-
anosyl, which was also supported by HSQC and HMBC data (Fig. 2). To determine the
absolute configuration of cymarose, the hydrolysate of 1 was analyzed by HPLC with

Helvetica Chimica Acta – Vol. 92 (2009)
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Fig. 1. Structures of the new compounds 1 – 8 isolated from Cynanchum wilfordii

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Table 1. 13C-NMR Data (100 MHz) of the Aglycone Moiety of Compounds 1 – 8 in CDCl3 (d in ppm)
1
2
3
4
5
6
7
8
C(1)
C(2)
C(3)
C(4)
38.7
28.8
77.8
38.7
38.8
29.0
78.0
38.9
38.9
28.9
78.0
38.9
38.8
28.9
78.0
38.8
38.8
28.9
77.9
38.8
38.8
28.9
77.9
38.8
36.9 36.9
28.7 28.7
76.6 76.6
37.8 37.8
C(5)
C(6)
C(7)
C(8)
140.2 140.1
117.8 118.2
140.3
118.0
34.3
74.4
43.7
37.2
24.4
72.9
58.5
88.2
33.4
32.0
91.6
9.7
140.5
118.1
34.3
74.4
43.7
37.2
24.4
72.9
58.6
88.2
33.4
32.0
91.7
9.7
140.4
117.9
34.3
74.3
43.7
37.2
24.4
72.9
58.5
88.2
33.4
32.1
91.6
9.7
140.4
117.9
34.3
74.3
43.7
37.2
24.4
71.5
58.5
88.2
33.4
32.1
91.6
9.7
141.2 141.2
118.3 118.3
40.8 40.7
209.5 209.5
55.2 55.1
42.7 42.6
25.4 25.3
70.8 70.8
34.1
74.2
43.2
37.2
24.8
73.3
56.4
87.8
32.9
32.5
87.2
10.4
18.3
75.6
14.9
34.4
74.4
43.5
37.1
25.2
73.8
56.3
88.0
33.1
32.4
87.3
10.3
18.4
74.6
15.2
C(9)
C(10)
C(11)
C(12)
C(13)
C(14)
C(15)
C(16)
C(17)
C(18)
C(19)
C(20)
C(21)
61.6
61.5
217.0 217.2
33.4 33.3
28.7 28.7
82.4 82.3
11.2
11.1
18.6
210.0
27.6
18.6
210.0
27.6
18.6
209.9
27.6
18.4
209.9
27.6
18.6 18.6
73.6 73.6
14.1 14.0
Substituent at C(12) Cina) Cin
p-OH-Bzb) p-OH-Bzb) p-OH-Bzb) Ikemc) Cin
Cin
C(1’)
C(2’)
C(3’)
C(4’)
C(5’/9’)
C(6’/8’)
C(7’)
166.2 167.0
118.6 118.6
144.2 145.3
134.0 134.3
127.9 128.5
128.7 129.1
130.2 130.6
165.4
121.9
132.0
115.5
161.0
115.5
132.0
165.4
121.9
132.0
115.5
161.1
115.5
132.0
165.4
121.7
131.9
115.5
161.2
115.5
131.9
166.0
113.1
167.0
38.3
21.1
21.0
166.8 166.8
117.6 117.6
145.3 145.3
134.0 133.9
127.9 127.9
128.9 128.8
130.6 130.5
Acf) Ac
16.7
Substituent at C(20) Nicd) Isovale)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
C(6’)
C(7’)
163.7 171.4
43.1
168.9 168.9
21.2
21.1
153.2
126.1
137.3
123.3
150.7
26.5
22.8
22.8
a) Cin ¼ cinnamoyl; b) p-OH-Bz ¼ p-hydroxybenzoyl; c) Ikem ¼ ikemaoyl; d) Nic ¼ nicotinoyl; e) Iso-
f
val ¼ isovaleryl; ) Ac ¼ acetyl.
refractive index (RI) and optical rotation (OR) detectors [5]. The OR detection
exhibited a positive signal for the cymarose, suggesting that the cymarose was of d-type.
The presence of b-d-oleandropyranosyl was confirmed by comparison with the
spectroscopic data in the literature [13]. The HMBC correlations from d(H) 3.52 – 3.55
(HꢀC(3) of aglycone) to d(C) 96.0 (C(1I)), and from d(H) 3.20 – 3.22 (HꢀC(4I)) to
d(C) 101.4 (C(1II)) led us to establish the linkage positions and sequences of the two

Helvetica Chimica Acta – Vol. 92 (2009)
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Table 2. 13C-NMR Data (100 MHz) of the Sugar Moieties of Compounds 1 – 8 in CDCl3 (d in ppm)
1
2
3
4
5
6
7
8
Moiety I
C(1I )
d-Cymc) d-Digitb) d-Digit d-Digit d-Digit d-Digit d-Cym
d-Cym
95.6
34.1
77.4
81.7
68.6
18.1
56.9
96.0
35.6
77.1
82.7
68.3
18.1
58.3
d-Oled)
101.4
35.2
80.5
75.3
71.4
95.9
37.3
66.7
83.0
68.0
18.3
95.9
37.2
66.8
82.8
68.1
18.3
95.7
37.1
66.5
82.8
67.9
18.1
95.6
37.1
66.7
82.7
67.9
18.1
95.7
37.1
66.5
82.7
67.9
18.1
95.6
34.1
77.2
81.7
68.7
18.1
57.4
C(2I )
C(3I )
C(4I )
C(5I )
C(6I )
MeOꢀC(3I )
Moiety II
C(1II )
d-Ole
100.5
35.5
80.5
75.3
71.9
d-Ole
100.5
36.0
78.8
81.6
72.0
18.5
56.5
l-Cym
97.2
31.1
d-Ole
100.2
35.8
78.4
81.8
d-Ole
100.1
35.7
78.3
81.8
d-Ole
100.2
35.9
78.5
81.7
l-Digina) l-Digin
100.8
31.5
73.7
74.3
66.7
18.0
55.5
d-Cym
99.2
34.2
77.0
81.7
68.9
18.2
56.9
l-Cym
98.5
30.9
74.7
71.9
65.5
17.4
56.3
100.7
31.4
73.7
74.0
66.6
17.3
55.4
d-Cym
99.0
32.8
76.9
71.9
70.9
17.9
57.8
C(2II )
C(3II )
C(4II )
C(5II )
71.7
71.7
71.8
C(6II )
17.9
56.4
18.1
56.6
18.1
56.4
l-Cym
97.5
31.5
18.1
56.2
l-Cym
97.5
31.6
18.0
56.3
l-Cym
97.5
31.6
MeOꢀC(3II )
Moiety III
C(1III
C(2III
C(3III
C(4III
C(5III
C(6III
)
)
)
)
)
)
75.1
72.3
65.3
17.8
56.5
72.3
76.7
64.1
17.7
56.9
d-Cym
94.4
33.4
77.2
72.3
70.8
18.2
57.2
72.7
76.8
63.8
17.9
56.8
d-Cym
94.5
35.0
77.2
81.9
68.7
18.1
57.6
l-Cym
98.3
30.9
74.7
72.0
65.5
18.0
56.4
72.7
76.9
63.9
17.7
57.8
d-Cym
94.6
35.2
77.1
81.7
68.6
18.2
56.8
l-Cym
98.2
30.9
74.7
72.0
65.6
18.1
56.2
MeOꢀC(3III
)
)
Moiety IV
C(1IV
C(2IV
C(3IV
C(4IV
C(5IV
C(6IV
)
)
)
)
)
)
MeOꢀC(3IV
Moiety V
C(1V )
C(2V )
C(3V )
C(4V )
C(5V )
C(6V )
MeOꢀC(3V )
a) Digin ¼ diginopyranosyl; b) Digit ¼ digitoxopyranosyl; c) Cym ¼ cymaropyranosyl; d) Ole ¼ olean-
dropyranosyl.
sugar units. Therefore, 1 was elucidated as 3-O-b-d-oleandropyranosyl-(1 ! 4)-b-d-
cymaropyranosyl gagaminin.
Wilfoside B (2) was isolated as a colorless, amorphous powder. The molecular
formula was established as C48H70O14 by HR-ESI-MS. The NMR data analysis of 2

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Fig. 2. Key HMBCs of compounds 1, 6, and 7
indicated that it possessed the same steroid part as 1 (Tables 1 and 3). The difference
was that there was an isovaleroyl group instead of a nicotinoyl group linked to C(20)
[5]. The linkage positions of the isovaleryl and cinnamoyl groups were ascertained by
the HMBC spectrum. The aglycone obtained upon acid hydrolysis of 2 was identified
as 12-O-cinnamoyl-20-O-isovaleroylsarcostin by comparsion with an authentic sample
[20].
1
The H-NMR spectrum of 2 showed two anomeric H-atom signals (d(H) 4.83, d,
J ¼ 8.7, and 4.52, d, J ¼ 9.9) for two b-linked sugar units (Table 4). On the basis of the
HSQC, HMBC, and acid hydrolysis experiments, as well as by comparison with the
literature [14], the two sugar moieties were identified as b-d-digitoxopyranosyl and b-
d-oleandropyranosyl. The sugar sequence was confirmed by the HMBC correlations

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Table 4. 1H-NMR Data (400 MHz) of the Sugar Moieties of Compounds 1 – 8 in CDCl3 (d in ppm,
J in Hz)
1
2
3
4
Moiety I
HꢀC(1I )
HꢀC(2I )
d-Cyma)
d-Digitb)
d-Digit
d-Digit
4.83 (d, J ¼ 8.1)
1.55 – 1.57 (m),
2.07 – 2.08 (m)
3.77 – 3.80 (m)
3.20 – 3.22 (m)
3.82 – 3.86 (m)
1.20 (d, J ¼ 6.3)
3.43 (s)
4.83 (d, J ¼ 8.7)
1.66 – 1.69 (m),
2.05 – 2.07 (m)
4.22 – 4.25 (m)
3.19 – 3.22 (m)
3.78 – 3.81 (m)
1.24 (d, J ¼ 6.0)
4.92 (d, J ¼ 8.7)
1.66 – 1.69 (m),
2.07 – 2.08 (m)
4.21 – 4.23 (m)
3.18 – 3.21 (m)
3.78 – 3.81 (m)
1.23 (d, J ¼ 6.0)
4.92 (d, J ¼ 8.7)
1.66 – 1.69 (m),
2.07 – 2.08 (m)
4.19 – 4.21 (m)
3.19 – 3.22 (m)
3.78 – 3.81 (m)
1.23 (d, J ¼ 6.0)
HꢀC(3I )
HꢀC(4I )
HꢀC(5I )
Me(6I )
MeOꢀC(3I )
Moiety II
HꢀC(1II )
HꢀC(2II )
d-Olec)
d-Ole
d-Ole
d-Ole
4.47 (d, J ¼ 7.8)
1.47 – 1.49 (m),
2.17 – 2.19 (m)
3.14 – 3.16 (m)
3.09 – 2.12 (m)
3.26 – 3.28 (m)
1.30 (d, J ¼ 6.2)
3.36 (s)
4.52 (d, J ¼ 9.9)
1.43 – 1.46 (m),
2.14 – 2.17 (m)
3.17 – 3.19 (m)
3.10 – 3.12 (m)
3.29 – 3.31 (m)
1.31 (d, J ¼ 6.0)
3.38 (s)
4.49 (d, J ¼ 8.7)
1.47 – 1.50 (m),
2.30 – 2.33 (m)
3.23 – 3.25 (m)
3.07 – 3.09 (m)
3.27 – 3.29 (m)
1.25 (d, J ¼ 6.0)
3.35 (s)
4.48 (d, J ¼ 8.7)
1.44 – 1.47 (m),
2.29 – 2.32 (m)
3.23 – 3.25 (m)
3.07 – 3.09 (m)
3.30 – 3.32 (m)
1.25 (d, J ¼ 6.0)
3.34 (s)
HꢀC(3II )
HꢀC(4II )
HꢀC(5II )
Me(6II )
MeOꢀC(3II )
Moiety III
l-Cym
l-Cym
HꢀC(1III
HꢀC(2III
)
)
4.84 (d, J ¼ 3.0)
1.70 – 1.73 (m),
2.20 – 2.23 (m)
3.55 – 2.58 (m)
3.27 – 3.29 (m)
4.05 – 4.07 (m)
1.22 (d, J ¼ 6.5)
3.36 (s)
4.84 (d, J ¼ 3.0)
1.72 – 1.76 (m),
2.20 – 2.23 (m)
3.67 – 3.70 (m)
3.57 – 3.59 (m)
4.25 – 4.28 (m)
1.22 (d, J ¼ 6.4)
3.38 (s)
HꢀC(3III
HꢀC(4III
HꢀC(5III
)
)
)
Me(6III
)
MeOꢀC(3III
)
Moiety IV
d-Cym
HꢀC(1IV
)
)
4.76 (d, J ¼ 8.7)
1.57 – 1.59 (m),
2.27 – 2.29 (m)
3.60 – 3.64 (m)
3.20 – 3.23 (m)
3.53 – 3.55 (m)
1.25 (d, J ¼ 6.4)
3.40 (s)
HꢀC(2IV
HꢀC(3IV
HꢀC(4IV
HꢀC(5IV
)
)
)
Me(6IV
)
MeOꢀC(3IV
Moiety V
HꢀC(1V )
HꢀC(2V )
)
HꢀC(3V )
HꢀC(4V )
HꢀC(5V )
Me(6V )
MeOꢀC(3V )
c
a) Cym: cymaropyranosyl; b) Digit: digitoxopyranosyl; ) Ole: oleandropyranosyl; d) Digin: diginopyranosyl.

Helvetica Chimica Acta – Vol. 92 (2009)
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Table 4 (cont.)
5
6
7
8
d-Digit
d-Digit
d-Cym
d-Cym
4.93 (d, J ¼ 9.9)
1.67 – 1.70 (m),
2.09 – 2.11 (m)
4.19 – 4.21 (m)
3.19 – 3.22 (m)
3.78 – 3.81 (m)
1.24 (d, J ¼ 6.0)
4.92 (d, J ¼ 9.9)
1.66 – 1.69 (m),
2.05 – 2.07 (m)
4.20 – 4.23 (m)
3.20 – 3.23 (m)
3.77 – 3.79 (m)
1.26 (d, J ¼ 6.0)
4.77 (d, J ¼ 8.1)
1.52 – 1.55 (m),
2.13 – 2.16 (m)
3.62 – 3.65 (m)
3.17 – 3.19 (m)
3.70 – 3.74 (m)
1.20 (d, J ¼ 6.3)
3.44 (s)
4.75 (d, J ¼ 8.1)
1.52 – 1.55 (m),
2.13 – 2.16 (m)
3.64 – 3.66 (m)
3.17 – 3.19 (m)
3.70 – 3.74 (m)
1.12 (d, J ¼ 6.3)
3.38 (s)
d-Ole
d-Ole
l-Digind)
l-Digin
4.48 (d, J ¼ 9.9)
1.44 – 1.47 (m),
2.30 – 2.33 (m)
3.27 – 3.29 (m)
3.07 – 3.09 (m)
3.27 – 3.29 (m)
1.22 (d, J ¼ 6.0)
3.32 (s)
4.50 (d, J ¼ 9.9)
1.48 – 1.51 (m),
2.30 – 2.33 (m)
3.25 – 3.27 (m)
3.07 – 3.10 (m)
3.30 – 3.33 (m)
1.24 (d, J ¼ 6.0)
3.38 (s)
4.93 (d, J ¼ 3.0)
1.79 – 1.81 (m),
1.99 – 2.01 (m)
3.55 – 3.57 (m)
3.81 – 3.84 (m)
3.89 – 3.91 (m)
1.21 (d, J ¼ 6.0)
3.44 (s)
4.93 (d, J ¼ 3.0)
1.79 – 1.81 (m),
1.99 – 2.01 (m)
3.56 – 3.58 (m)
3.81 – 3.84 (m)
3.90 – 3.92 (m)
1.19 (d, J ¼ 6.0)
3.40 (s)
l-Cym
l-Cym
d-Cym
d-Cym
4.82 (d, J ¼ 3.0)
1.72 – 1.75 (m),
2.20 – 2.22 (m)
3.72 – 3.75 (m)
3.55 – 3.57 (m)
4.26 – 4.29 (m)
1.18 (d, J ¼ 6.5)
3.46 (s)
4.82 (d, J ¼ 3.0)
1.70 – 1.74 (m),
2.21 – 2.24 (m)
3.52 – 3.55 (m)
3.70 – 3.74 (m)
4.24 – 4.28 (m)
1.20 (d, J ¼ 6.5)
3.46 (s)
4.75 (d, J ¼ 8.7)
1.72 – 1.76 (m),
2.27 – 2.29 (m)
3.74 – 3.76 (m)
3.25 – 3.27 (m)
3.82 – 3.85 (m)
1.14 (d, J ¼ 6.5)
3.39 (s)
4.64 (d, J ¼ 8.1)
1.60 – 1.63 (m),
2.35 – 2.37 (m)
3.64 – 3.67 (m)
3.25 – 3.27 (m)
3.52 – 3.56 (m)
1.26 (d, J ¼ 6.5)
3.38 (s)
d-Cym
d-Cym
l-Cym
4.86, (d, J ¼ 8.7)
1.60 – 1.63 (m),
2.14 – 2.17 (m)
3.70 – 3.71 (m)
3.23 – 3.25 (m)
3.83 – 3.85 (m)
1.22 (d, J ¼ 6.4)
3.35 (s)
4.85, (d, J ¼ 8.7)
1.60 – 1.63 (m),
2.14 – 2.16 (m)
3.67 – 3.69 (m)
3.23 – 3.25 (m)
3.82 – 3.84 (m)
1.24 (d, J ¼ 6.0)
3.35 (s)
4.76 (d, J ¼ 3.0)
1.70 – 1.72 (m),
2.23 – 2.25 (m)
3.55 – 3.57 (m)
3.23 – 3.25 (m)
4.00 – 4.02 (m)
1.20 (d, J ¼ 6.4)
3.40 (s)
l-Cym
l-Cym
4.80 (d, J ¼ 3.0)
1.73 – 1.76 (m),
2.27 – 2.30 (m)
3.57 – 3.60 (m)
3.27 – 3.29 (m)
4.03 – 4.05 (m)
1.19 (d, J ¼ 6.4)
3.38 (s)
4.79 (d, J ¼ 3.0)
1.70 – 1.73 (m),
2.27 – 2.30 (m)
3.57 – 3.60 (m)
3.27 – 3.29 (m)
4.03 – 4.05 (m)
1.21 (d, J ¼ 6.4)
3.43 (s)

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Helvetica Chimica Acta – Vol. 92 (2009)
between i) HꢀC(3) (d(H) 3.52 – 3.55, m) of the aglycone and C(1I) (d(C) 95.9) of the
b-d-digitoxopyranosyl moiety; ii) HꢀC(4I) (d(H) 3.19 – 3.22, m) of the b-d-digit-
oxopyranosyl moiety and C(1II) (d(H) 100.5) of the b-d-oleandropyranosyl moiety
[21]. Therefore, 2 was elucidated as 3-O-b-d-oleandropyranosyl-(1 ! 4)-b-d-digitoxo-
pyranosyl 12-O-cinnamoyl-20-O-isovaleroylsarcostin.
Wilfoside C (3) was isolated as a colorless, amorphous powder. The molecular
formula was determined as C48H70O17 by HR-ESI-MS. The aglycone of 3 was identified
as qingyangshengenin by comparing its spectroscopic data with those of known C21
sterodial aglycones [10] as well as upon acid hydrolysis.
Comparing the structure of the sugar moiety of 3 with that of 2, there was one more
sugar unit linked with the b-d-oleandropyranosyl. The coupling constant of the
anomeric H-atom signal of the third sugar (d(H) 4.84, d, J ¼ 3.0) indicated an a-
configuration. On acid hydrolysis, 3 afforded cymarose, digitoxose, and oleandrose as
component sugars. According to the literatures [4 – 22], the chemical shift of C(2) is
< 33.0 ppm in an l-cymaropyranosyl unit and > 35.0 ppm in a d-cymaropyranosyl unit
[10]. The 13C-NMR signals of the third sugar unit assigned by HSQC and HMBC
analyses, and in particular the resonance of C(2III) at d(C) 31.1, indicated the presence
of one a-l-cymaropyranosyl moiety. The OR detection exhibited a negative signal for
the cymarose, which further confirmed the l-configuration of cymarose. Following the
methodology described above, the sequence of the sugar moieties was assigned from
HSQC and HMBC analysis using the well-defined anomeric H-atoms as starting
signals. Therefore, 3 was elucidated as 3-O-a-l-cymaropyranosyl-(1 ! 4)-b-d-olean-
dropyranosyl-(1 ! 4)-b-d-digitoxopyranosyl qingyangshengenin.
Wilfoside D (4) was isolated as a colorless, amorphous powder. The positive HR-
ESI-MS gave the molecular formula C55H82O20. Compound 4 was determined to
possess the same aglycone as 3 from the NMR data and the acid hydrolysis experiment,
and the structure of the sugar moiety was corresponding to that of 3 except that one
more sugar unit was linked to the a-l-cymaropyranosyl moiety. The coupling constant
of the anomeric H-atom signal in the fourth sugar (d(H) 4.76, d, J ¼ 8.7) indicated a b-
configuration. The chemical shifts and TLC behavior suggested that the sugar was a b-
d-cymaropyranosyl moiety. Similarly as what was carried out on 3, HPLC analysis
revealed that both d- and l-cymaroses occurred in 4 since OR of the cymarose was
detected to be zero. Following the same methodology described above, the sequence of
the sugar moieties of 4 was assigned. Therefore, 4 was elucidated as 3-O-b-d-
cymaropyranosyl-(1 ! 4)-a-l-cymaropyranosyl-(1! 4)-b-d-oleandropyranosyl-(1! 4)-
b-d-digitoxopyranosyl qingyangshengenin.
Wilfoside E (5) was isolated as a colorless, amorphous powder. Based on the HR-
ESI-MS data, the molecular formula was established as C62H94O23. The NMR of 5 were
similar to 4, indicating the same aglycone, and the sugar moiety in 5 was corresponding
to that of 4 except that one more sugar unit was linked to the b-d-cymaropyranosyl
1
moiety. The sugar unit was identified as a-l-cymaropyranosyl by the H-, 13C-NMR,
TLC, and HPLC data. An extensive study of the HMBC experiment enabled to
establish the linkage positions and sequence of the sugar moieties of 5. Therefore, 5 was
elucidated as 3-O-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-
cymaropyranosyl-(1 ! 4)-b-d-oleandropyranosyl-(1 ! 4)-b-d-digitoxo-
pyranosyl qingyangshengenin.

Helvetica Chimica Acta – Vol. 92 (2009)
2669
Wilfoside F (6) was isolated as a colorless, amorphous powder. The molecular
formula was obtained as C62H100O22 by HR-ESI-MS. The NMR data analysis of 6
indicated that it possessed the same steroid part and sugar chain as those of 5. The
difference was that there was an ikemaoyl group instead of a p-hydroxybenzoyl
substituent at C(12). The aglycone of 6 was determined to be caudatin (Table 1) by
comparing the NMR data with those of known C21 steroidal aglycones [18]. The
structure of 6 was further confirmed by acid hydrolysis, HSQC, HMBC (Fig. 2), and
1H,1H-COSY experiments to be 3-O-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyrano-
syl-(1 ! 4)-a-l-cymaropyranosyl-(1 ! 4)-b-d-oleandropyranosyl-(1 ! 4)-b-d-digitoxo-
pyranosyl caudatin.
Wilfoside G (7) was isolated as a colorless, amorphous powder. The molecular
formula was established as C60H88O20 by HR-ESI-MS. When comparing the NMR data
of 7 with those of wilfoside KIN (9) [12], the two O-bearing quaternary C-atom signals
C(8) and C(14) (d(C) 74.2 and 88.1) of 9 replaced by two CO group (d(C) 209.5 and
217.0) signals in 7, suggesting the oxidation of the two OH groups. Oxidative cleavage of
the 8b,14b-diol of 9 (30 mg) with lead tetraacetate yielded 24.5 mg of diketone product
(9a) [17] (Scheme). Lead tetraacetate is widely used as an oxidizing agent in organic
chemistry for 1,2-diol cleavage to obtain ketone products. The reaction should occur in
1
organic solvent such as benzene. The H-NMR spectra of 9 and 9a (Table 5) showed
different chemical shifts of HꢀC(12) (d(H) 5.20 – 5.24, m, in 9a vs. d(H) 4.71 – 4.73, m,
in 9) and the HOꢀC(8/14) (d(H) 4.36, 4.19 in 9) were disappeared in 9a. 9a was
elucidated as 3-O-a-l-cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-
diginopyranosyl-(1 ! 4)-b-d-cymaropyranosyl kidjoranin-8,14-dione.
Scheme. Lead Tetraacetate Oxidation of 9
The close resemblance of the NMR data of 7 and 9a indicated that they possessed
the same steroid part and sugar chain, but that an acetyl substituent was linked to C(20)
in 7, which was confirmed from the HMBC correlation between C(1’Ac) (d(C) 168.9)
and HꢀC(20) (d(H) 4.82 – 4.85). A Me group (d(C) 14.1) was assigned to be linked to

2670
Helvetica Chimica Acta – Vol. 92 (2009)
Table 5. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) Data of Compounds 9 and 9a in CDCl3 (d in
ppm, J in Hz)
9
9a
d(H)
d(C)
d(H)
d(C)
CH2(1)
CH2(2)
1.08 – 1.11 (m), 1.80 – 1.83 (m)
1.58 – 1.62 (m), 1.89 – 1.91 (m)
3.60 – 3.63 (m)
38.8 1.14 – 1.16 (m), 1.77 – 1.79 (m)
28.9 1.46 – 1.48 (m), 1.93 – 1.95 (m)
72.7 3.50 – 3.53 (m)
38.8 2.07 – 2.09 (m), 2.39 – 2.41 (m)
140.5
117.8 5.32 (br. s)
34.3 2.60 – 2.64 (m), 3.01 – 3.03 (m)
37.1
29.0
76.9
38.1
141.5
118.7
41.0
HꢀC(3)
CH2(4)
2.23 – 2.27 (m), 2.35 – 2.37 (m)
C(5)
HꢀC(6)
5.38 (br. s)
2.13 – 2.17 (m)
CH2(7)
C(8)
74.2
209.7
55.3
42.9
23.4
71.8
HꢀC(9)
1.47 – 1.51 (m)
43.6 2.27 – 2.29 (m)
37.2
24.2 1.42 – 1.45 (m), 1.85 – 1.87 (m)
72.1 5.20 – 5.24 (m)
C(10)
CH2(11)
HꢀC(12)
C(13)
1.77 – 1.79 (m), 2.04 – 2.06 (m)
4.71 – 4.73 (m)
58.0
61.4
C(14)
88.1
214.0
34.1
29.5
85.7
14.1
18.9
209.5
27.1
CH2(15)
CH2(16)
C(17)
Me(18)
Me(19)
2.00 – 2.01 (m)
1.90 – 1.91 (m), 2.83 – 2.86 (m)
33.2 2.43 – 2.45 (m), 2.70 – 2.74 (m)
32.0 1.95 – 1.97 (m), 2.13 – 2.15 (m)
91.5
9.5 1.36 (s)
18.5 1.07 (s)
209.5
1.55 (s)
1.09 (s)
C(20)
Me(21)
2.08 (s)
27.5 2.01 (s)
Cin at C(12)
C(1’)
HꢀC(2’)
HꢀC(3’)
C(4’)
HꢀC(5’/9’)
HꢀC(6’/8’)
HꢀC(7’)
Moiety I: d-Cym
HꢀC(1I )
HꢀC(2I )
HꢀC(3I )
HꢀC(4I )
HꢀC(5I )
Me(6I )
165.8
166.4
116.8
146.3
134.1
128.3
129.1
130.8
6.08 (d, J ¼ 15.9)
7.35 (d, J ¼ 17.7)
117.6 6.08 (d, J ¼ 15.9)
145.5 7.35 (d, J ¼ 17.7)
134.2
128.2 7.18 – 7.21 (m)
128.9 7.28 – 7.31 (m)
130.5 7.30 – 7.33 (m)
7.18 – 7.21 (m)
7.28 – 7.31 (m)
7.30 – 7.33 (m)
4.81 (d, J ¼ 8.7)
1.55 – 1.58 (m), 2.10 – 2.13 (m)
3.65 – 3.68 (m)
3.20 – 3.22 (m)
3.90 – 3.93 (m)
1.20 (overlap)
95.7 4.83 (d, J ¼ 8.7)
34.3 1.57 – 1.59 (m), 2.10 – 2.13 (m)
77.2 3.67 – 3.69 (m)
81.9 3.20 – 3.23 (m)
69.1 3.87 – 3.89 (m)
18.3 1.22 (overlap)
57.1 3.42 (s)
95.9
34.5
77.2
81.9
68.9
18.3
57.1
MeOꢀC(3I )
Moiety II: l-Digin
HꢀC(1II )
HꢀC(2II )
HꢀC(3II )
HꢀC(4II )
HꢀC(5II )
Me(6II )
3.38 (s)
4.96 (d, J ¼ 3.0)
1.70 – 1.73 (m), 1.95 – 1.97 (m)
3.73 – 3.75 (m)
3.85 – 3.88 (m)
3.94 – 3.97 (m)
1.20 (overlap)
100.9 4.98 (d, J ¼ 3.0)
31.1 1.73 – 1.76 (m), 1.93 – 1.96 (m)
73.9 3.72 – 3.75 (m)
74.5 3.87 – 3.89 (m)
66.8 3.96 – 3.99 (m)
18.2 1.22 (overlap)
55.6 3.41 (s)
100.9
31.2
74.0
74.5
66.9
18.2
55.7
MeOꢀC(3II )
3.40 (s)
Moiety III: d-Cym
HꢀC(1III
)
4.76 (d, J ¼ 9.0)
99.4 4.75 (d, J ¼ 9.0)
99.4

Helvetica Chimica Acta – Vol. 92 (2009)
2671
Table 5 (cont.)
9
9a
d(H)
d(C)
d(H)
d(C)
HꢀC(2III
HꢀC(3III
HꢀC(4III
HꢀC(5III
)
)
)
)
1.70 – 1.73 (m), 2.21 – 2.23 (m)
3.82 – 3.85 (m)
3.26 – 2.29 (m)
3.86 – 3.88 (m)
1.20 (overlap)
34.3
77.2
81.9
69.1
18.2
57.5
1.70 – 1.71 (m), 2.23 – 2.25 (m)
3.82 – 3.85 (m)
3.26 – 2.29 (m)
3.86 – 3.89 (m)
1.22 (overlap)
34.5
77.3
81.9
69.1
18.2
57.6
Me(6III
)
MeOꢀC(3III
)
3.45 (s)
3.46 (s)
Moiety IV: l-Cym
HꢀC(1IV
HꢀC(2IV
HꢀC(3IV
HꢀC(4IV
HꢀC(5IV
)
)
)
)
)
4.84 (d, J ¼ 3.0)
1.64 – 1.66 (m), 2.17 – 2.19 (m)
3.61 – 3.62 (m)
3.23 – 3.25 (m)
4.01 – 4.03 (m)
1.20 (overlap)
98.6
31.7
74.9
72.1
65.7
17.6
56.4
4.83 (d, J ¼ 3.0)
1.64 – 1.67 (m), 2.17 – 2.19 (m)
3.61 – 3.63 (m)
3.23 – 2.26 (m)
4.01 – 4.04 (m)
1.22 (overlap)
98.6
31.7
74.9
72.2
65.8
17.6
56.5
Me(6IV
)
MeOꢀC(3IV
)
3.37 (s)
3.39 (s)
Cin ¼ cinnamoyl; Digin ¼ diginopyranosyl; Cym ¼ cymaropyranosyl; Ole ¼ oleandropyranosyl
C(20) from the cross-peaks of d(H) 4.82 – 4.85 (HꢀC(20)) to d(C) 14.1 in the HMBC
spectrum. Two CO C-atoms were identified as C(8) and C(14) based on the HMBC
correlations between C(8) (d(C) 209.5) and HꢀC(6) (d(H) 5.32), CH2(7) (d(H) 2.60 –
2.63, 3.01–3.04), HꢀC(9) (d(H) 2.26–2.30), and CH2(11) (d(H) 1.43 –1.45, 1.85 –1.87),
and between C(14) (d(C) 217.0) and CH2(15) (d(H) 2.43 – 2.45, 2.70 – 2.73) and
CH2(16) (d(H) 1.95 – 1.97, 2.13 – 2.15) [4][23][24]. There were key HMBCs observed
between C(5) (d(C) 141.2) and CH2(7), between C(2) (d(C) 28.7) and CH2(4) (d(H)
2.07 – 2.09, 2.39 – 2.41), between C(9) (d(C) 55.2) and CH2(1) (d(H) 1.13 – 1.17, 1.76 –
1.80), and between C(10) (d(C) 42.7) and CH2(4) (Fig. 2). Therefore, the aglycone of 7
was determined to be 12-O-cinnamoyl-20-O-acetyl-8,14-secosarcostin-8,14-dione.
Compared with NMR data in the literature [23 – 25], 7 was elucidated as 3-O-a-l-
cymaropyranosyl-(1 ! 4)-b-d-cymaropyranosyl-(1 ! 4)-a-l-diginopyranosyl-(1 ! 4)-
b-d-cymaropyranosyl 12-O-cinnamoyl-20-O-acetyl-8,14-secosarcostin-8,14-dione.
Wilfoside H (8) was isolated as a colorless, amorphous powder. The molecular
formula was established as C53H77O17 by HR-ESI-MS. The similar NMR data of 7 and 8
indicated that they possessed the same aglycone. The chemical shifts of C(4III) (d(C)
71.9 in 8 vs. d(C) 81.7 in 7) of the third sugar unit indicated one less b-d-
cymaropyranosyl unit than in 7. On acid hydrolysis, 8 afforded cymarose, diginose,
and oleandrose as sugar components. Therefore, 8 was elucidated as 3-O-b-d-
cymaropyranosyl-(1 ! 4)-a-l-diginopyranosyl-(1 ! 4)-b-d-cymaropyranosyl 12-O-cin-
namoyl-20-O-acetyl-8,14-secosarcostin-8,14-dione.
Experimental Part
General. All solvents used were of anal. grade (Shanghai Chemical Plant). TLC: Silica gel GF 254
(Yantai Huiyou Inc.). Column chromatography (CC): silica gel H (SiO2; 200 – 300 mesh; Qingdao

2672
Helvetica Chimica Acta – Vol. 92 (2009)
Marine Chemical Ltd.), Sephadex LH-20 (25 – 100 mm; Pharmacia Fine Chemicals), MCI gel CHP 20P
(high porous polymer, 75 – 150 mm; Mitsubishi Chemical Ind.), and RP-18 (20 – 45 mm; Fuji Silysia
Chemical Ltd.). HPLC Separations: JASCO PU-2080 HPLC system, equipped with RI (G1632A 1100
RID) and OR (Shodex OR-2) detectors. Optical rotations: in MeOH soln.; Perkin-Elmer PE-241
polarimeter; JASCO DIP-370 digital polarimeter; in a 0.5 dm length cell. UV Spectra: Varian CARY 300
Bio spectrometer; lmax in nm (log e). IR Spectra: Perkin-Elmer 16-PC FT-IR spectrophotometer; in
cmꢀ1. 1H- (400 MHz) and 13C-NMR (100 MHz) Spectra: Bruker AMX-400 spectrometer; d in ppm, J in
Hz, with Me4Si as internal standard. HR-ESI-MS: Bruker Daltonics FTMS APEX III mass spectrometer
in m/z.
Plant Material. The roots of C. wilfordii were collected in March 2007 in Hebei Province, P. R. China.
A voucher specimen (No. 200706) was deposited with the Shanghai Research Center for Modernization
of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai.
Extraction and Isolation. The dried roots of C. wilfordii (5 kg) were extracted three times with 95%
EtOH (40 l) under reflux for 2 h, which afforded a dark residue (600 g) after evaporation. The residue
was partitioned between AcOEt (3 l) and H2O (3 l). The org. layer (200 g after evaporation) was
subjected to CC (SiO2, petroleum ether (PE)/acetone 10 :1 – 1:1, and 100% acetone) to give five
fractions (Frs. A – E). Fr. B was subjected to CC (MCI gel; gradient MeOH/H2O 40 :60 ! 95 :5), then to
CC (SiO2; CHCl3/acetone 4 :1), to CC (RP-18; MeCN/H2O 50 :50), and to CC (RP-18; MeOH/H2O
70 :30) to yield 1 (28 mg), 7 (25 mg), 8 (26 mg), and 9 (70 mg). Fr. C was subjected to CC (MCI gel,
gradient MeOH/H2O 40 :60 ! 95 :5), then to CC (Sephadex LH-20; CHCl3/MeOH 1:1), and to CC
(RP-18; MeCN/H2O 45 :55) to yield 2 (25 mg) and 6 (16 mg). Fr. D (5.0 g) was subjected to CC (RP-18;
MeCN/H2O 30 :70) to yield 3 (11 mg), 4 (14 mg), and 5 (30 mg).
Acid Hydrolysis. A soln. of 1 – 8 (each 6 mg) in MeOH (5 ml) was treated separately with 0.05m HCl
(dioxane/H2O 1:1, 1 ml) at 608 for 1.5 h. After dioxane was removed, the soln. was extracted with 2 ml of
AcOEt for three times. The aq. layer was neutralized by NaOH (1m) and concentrated under reduced
pressure to give the sugar fraction. The identification of the monosaccharides in the hydrolysates of each
compound was confirmed by TLC comparison with authentic sugars: digitoxose was detected from 2, 3, 4,
and 5; diginose was detected from 7 and 8; cymarose was detected from 1, 3, 4, 5, 7, and 8; oleandrose was
detected from 1 and 2. The Rf values of digitoxose, diginose, cymarose, and oleandrose were 0.21, 0.37,
0.48, and 0.42 (CHCl3/MeOH 9 :1), 0.28, 0.39, 0.51, and 0.48 (CH2Cl2/EtOH 9 :1), and 0.09, 0.19, 0.23, and
0.20 (PE/Me2CO 3 :2), resp. [5]. The AcOEt-soluble portion was concentrated to dryness to give the
crude aglycone. The crude aglycones were identified by TLC comparison with authentic samples (the
aglycone of 6 was caudatin, the aglycone of 1 was gagaminin, the aglycone of 2 was 12-O-cinnamoyl-20-
O-isovaleroylsarcostin and the aglycone of 3, 4, and 5 was qingyangshengenin).
Determination of the Absolute Configuration of the Monosaccharides. The sugar fractions were
obtained from the aq. layer by acid hydrolysis as described above. The absolute configurations were
analyzed by HPLC [8][26][27]. Identification of d-cymarose and l-diginose in each sugar fraction was
performed by comparing their retention time and optical rotation polarity with those of authentic
samples: d-cymarose (tR 14.687 min, pos. polarity) and l-diginose (tR 15.794 min, neg. polarity; HPLC
conditions: column, Shodex Asahipak GS-220 HQ, 300 ꢁ 7.5 mm i.d.; flow rate, 0.5 ml/min; column
temp., 408; solvent H2O; detection with RI (Refractive Index Detector G1632A 1100 RID) and OR
(Optical Rotation Detector Shodex OR-2) detectors. Cymarose (a mixture of d- and l-form in the ratio
1:1) was detected from 4 and 6. d-Cymarose was detected from 1, 7, and 8, and l-cymarose was detected
from 3, 5, and 6.
Pb(OAc)4 Oxidation of 9. To a soln. of 9 (30 mg) in 4 ml of benzene, 80 mg of lead tetraacetate was
added, and the mixture was stirred for 30 min at r.t. H2O was added, and the mixture was then extracted
with Et2O. The Et2O soln. was washed successively with 5% NaHCO3 soln. and H2O, and dried over
MgSO4. After removal of the solvent and purification of the residue, 24.5 mg of diketone 9a was obtained
[17]. (1R)-1-[(1R,2S)-2-Acetyl-2-hydroxy-1-methyl-5-oxocyclopentyl]-2-[(6S,8aR)-6-{[2,6-dideoxy-3-O-
methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-di-
deoxy-3-O-methyl-a-l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl]-
oxy}-8a-methyl-2-oxo-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl]ethyl (2E)-3-phenylprop-2-enoate (9a).
Colorless, amorphous powder. [a]2D0 ¼ ꢀ53 (c ¼ 0.3, MeOH). UV (MeOH): 218 (4.20), 223 (4.16), 282

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1
(4.40). IR (KBr): 3440, 2935, 1733, 1715, 1635, 1450, 1734, 1252, 1163, 1100, 1057. H-NMR: Table 5.
13C-NMR: Table 5. HR-ESI-MS: 1107.5468 ([M þ Na]þ, C53H77NaO1þ7 ; calc. 1107.5454).
Wilfoside A (¼(3b,12b,14b,17a)-3-{[2,6-Dideoxy-4-O-(2,6-dideoxy-3-O-methyl-b-d-arabino-hexo-
pyranosyl)-3-O-methyl-b-d-ribo-hexopyranosyl]oxy}-8,14,17-trihydroxy-12-{[(2E)-3-phenylprop-2-
enoyl]oxy}pregn-5-en-20-yl Pyridine-3-carboxylate; 1). Colorless, amorphous powder. [a]2D0 ¼ þ118 (c ¼
1
0.3, MeOH). UV (MeOH): 217 (4.44), 280 (4.37). IR (KBr): 3438, 2933, 1716, 1637, 1450. H-NMR:
Tables 3 and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 906.4648 ([M þ H]þ, C50H68NO1þ4 ; calc.
906.4640).
Wilfoside B (¼(3b,12b,14b,17a)-3-{[2,6-Dideoxy-4-O-(2,6-dideoxy-3-O-methyl-b-d-arabino-hexo-
pyranosyl)-b-d-ribo-hexopyranosyl]oxy}-8,14,17-trihydroxy-12-{[(2E)-3-phenylprop-2-enoyl]oxy}pregn-
5-en-20-yl 3-Methylbutanoate; 2). Colorless, amorphous powder. [a]2D0 ¼ þ41 (c ¼ 0.3, MeOH). UV
(MeOH): 205 (4.09), 217 (3.73), 278 (4.69). IR (KBr): 3446, 2933, 1710, 1637, 1450. 1H-NMR: Tables 3
and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 893.4667 ([M þ Na]þ, C48H70NaO1þ4 ; calc. 893.4663).
Wilfoside C (¼(3b,12b,14b,17a)-3-{[2,6-Dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-
dideoxy-3-O-methyl-b-d-arabino-hexopyranosyl-(1 ! 4)-2,6-dideoxy-b-d-ribo-hexopyranosyl]oxy}-
8,14,17-trihydroxy-20-oxopregn-5-en-12-yl 4-Hydroxybenzoate; 3). Colorless, amorphous powder.
[a]2D0 ¼ ꢀ27 (c ¼ 0.3, MeOH). UV (MeOH): 212 (3.40), 255 (4.14). IR (KBr): 3446, 2933, 1710, 1610,
1450. 1H-NMR: Tables 3 and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 941.4524 ([M þ Na]þ,
C48H70NaOþ17 ; calc. 941.4511).
Wilfoside D (¼(3a,12b,14b,17a)-3-{[2,6-Dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-
dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-arabino-hexopyrano-
syl-(1 ! 4)-2,6-dideoxy-b-d-ribo-hexopyranosyl]oxy}-8,14,17-trihydroxy-20-oxopregn-5-en-12-yl 4-Hy-
droxybenzoate; 4). Colorless, amorphous powder. [a]2D0 ¼ ꢀ36 (c ¼ 0.3, MeOH). UV (MeOH): 212
(3.44), 255 (4.18). IR (KBr): 3453, 2933, 1712, 1610, 1450. 1H-NMR Tables 3 and 4. 13C-NMR: Tables 1
and 2. HR-ESI-MS: 1085.5305 ([M þ Na]þ, C55H82NaO2þ0 ; calc. 1085.5297).
Wilfoside E (¼(3a,12b,14b,17a)-3-{[2,6-Dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-
dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-
(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-arabino-hexopyranosyl-(1 ! 4)-2,6-dideoxy-b-d-ribo-hexopyrano-
syl]oxy}-8,14,17-trihydroxy-20-oxopregn-5-en-12-yl 4-Hydroxybenzoate; 5). Colorless, amorphous pow-
der. [a]2D0 ¼ ꢀ56 (c ¼ 0.3, MeOH). UV (MeOH): 212 (3.41), 255 (4.15). IR (KBr): 3448, 2933, 1712,
1
1610, 1450. H-NMR Tables 3 and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 1229.6079 ([M þ Na]þ,
C62H94NaOþ23 ; calc. 1229.6084).
Wilfoside F (¼(3a,12b,14b,17a)-3-{[2,6-Dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-
dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-
(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-arabino-hexopyranosyl-(1 ! 4)-2,6-dideoxy-b-d-ribo-hexopyrano-
syl]oxy}-8,14,17-trihydroxy-20-oxopregn-5-en-12-yl (2E)-3,4-Dimethylpent-2-enoate; 6). Colorless,
amorphous powder. [a]2D0 ¼ ꢀ58 (c ¼ 0.3, MeOH). UV (MeOH): 225 (0.79), 272 (4.76). IR (KBr):
1
3446, 1714, 1641. H-NMR: Tables 3 and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 1219.6611 ([M þ
Na]þ, C62H100NaO2þ2 ; calc. 1219.6604).
Wilfoside G (¼(1R)-1-{(1R,2S)-2-[(1S)-1-(Acetyloxy)ethyl]-2-hydroxy-1-methyl-5-oxocyclopentyl}-
2-[(1R,6S,8aR)-6-{[2,6-dideoxy-3-O-methyl-a-l-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-
d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-
O-methyl-b-d-ribo-hexopyranosyl]oxy}-8a-methyl-2-oxo-1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl]ethyl
(2E)-3-Phenylprop-2-enoate; 7). Colorless, amorphous powder. [a]2D0 ¼ ꢀ58 (c ¼ 0.3, MeOH). UV
(MeOH): 217 (2.95), 272 (3.61), 278 (3.64). IR (KBr): 3448, 2933, 1716, 1637, 1450. 1H-NMR: Tables 3
and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 1151.5781 ([M þ Na]þ, C60H88NaO2þ0 ; calc. 1151.5767).
Wilfoside H (¼(1R)-1-{(1R,2S)-2-[(1S)-1-(Acetyloxy)ethyl]-2-hydroxy-1-methyl-5-oxocyclopentyl}-
2-[(1R,6S,8aR)-6-{[2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-a-
l-lyxo-hexopyranosyl-(1 ! 4)-2,6-dideoxy-3-O-methyl-b-d-ribo-hexopyranosyl]oxy}-8a-methyl-2-oxo-
1,2,3,5,6,7,8,8a-octahydronaphthalen-1-yl]ethyl (2E)-3-Phenylprop-2-enoate; 8). Colorless, amorphous
powder. [a]2D0 ¼ ꢀ19 (c ¼ 0.3, MeOH). UV (MeOH): 218 (2.94), 273 (3.62), 278 (3.63). IR (KBr): 3448,
2933, 1733, 1714, 1637, 1450. 1H-NMR: Tables 3 and 4. 13C-NMR: Tables 1 and 2. HR-ESI-MS: 985.5150
([M þ H]þ, C53H77O1þ7 ; calc. 985.5161).

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Received February 20, 2009