Cynanosides A-J, ten novel pregnane glycosides from Cynanchum atratum

Article abstract of DOI:10.1016/j.tet.2005.04.015

Four novel 13,14:14,15-diseco-18-nor-pregnane-type steroidal glycosides, cynanosides A-D (1-4), and six novel 13,14:14,15-diseco-pregnane-type steroidal glycosides, cynanosides E-J (5-10) were isolated from the roots of Cynanchum atratum, together with one known compound, cynatratoside F (11). Their structures including the absolute stereochemistry were determined on the basis of spectroscopic analysis and chemical evidence, with combination of the modified Mosher method, the exciton chirality method and chemical transformations.

Full text of DOI:10.1016/j.tet.2005.04.015

Tetrahedron 61 (2005) 5797–5811
Cynanosides A–J, ten novel pregnane glycosides from
Cynanchum atratum
Hong Bai,a Wei Li,a Kazuo Koike,a Tadaaki Satou,a Yingjie Chenb and Tamotsu Nikaidoa,
*
aFaculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
bSchool of Chinese Traditional Medicine, Shenyang Pharmaceutical University, Shenyang 110016, China
Received 8 March 2005; revised 11 April 2005; accepted 11 April 2005
Available online 10 May 2005
Abstract—Four novel 13,14:14,15-diseco-18-nor-pregnane-type steroidal glycosides, cynanosides A–D (1–4), and six novel 13,14:14,15-
diseco-pregnane-type steroidal glycosides, cynanosides E–J (5–10) were isolated from the roots of Cynanchum atratum, together with one
known compound, cynatratoside F (11). Their structures including the absolute stereochemistry were determined on the basis of
spectroscopic analysis and chemical evidence, with combination of the modified Mosher method, the exciton chirality method and chemical
transformations.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
chemistry of the new compounds on the basis of spectro-
scopic analysis and chemical evidence, with combination of
the modified Mosher method, the exciton chirality method
and chemical transformations.
Pregnanes and their glycosides have established themselves
as an important class of biologically active compounds.
Pharmacological studies in recent years, have shown that
pregnane glycosides possess antitumor,1 bone resorbing2
and anti-fungal activities.3 A number of such compounds
have been isolated from the plants, especially the
2. Results and discussion
Asclepiadaceae
family.4
Cynanchum
atratum
Cynanoside A (1) was obtained as an amorphous powder.
The molecular formula was determined to be C41H62O15 by
HRFABMS (m/z 817.4023 [MCNa]C, calcd for 817.3986).
Its IR absorptions at 3423 and 1737 cmK1 suggested the
presence of hydroxyl and carbonyl groups. The 13C NMR
data (Table 1) in combination with analysis of the DEPT
and HMQC spectra revealed 41 carbon signals due to six
quaternary carbons, 19 methines, eight methylenes and
eight methyls, of which 20 were assigned to the aglycon part
including two carbonyl carbons at d 196.0 and 178.9, and
the remaining 21 were ascribed to the sugar moiety
(Asclepiadaceae) is a perennial herb native to east Asia
and its roots, commonly called ‘Bai Wei’ in Chinese, have
been used in traditional medicine for the treatment of hectic
fevers, acute urinary infection and abscesses.5 Previous
phytochemical studies on this species have reported the
occurrence of 14,15-seco-pregnane type and 13,14:14,15-
diseco-pregnane type glycosides.6 As our current interest in
the chemistry of biologically active and structurally unique
natural products, we also carried out a chemical investi-
gation on C. atratum, which resulted in the isolation of ten
new glycosides, cynanosides A–J (1–10), together with one
known compound cynatratoside F (11)6b (Fig. 1). Among
them, 1–3 and 4, with two new aglycons, named
cynanogenin A (12) and cynanogenin B (13), have a novel
13,14:14,15-diseco-18-nor-pregnane skeleton, while 5–8
and 9–11, with two known aglycons, hancopregnane (14)
and glaucogenin A (15), possess a 13,14:14,15-diseco-
pregnane skeleton. This paper describes the isolation and
structural elucidation including the absolute stereo-
1
(including three methoxy groups). The H NMR spectrum
of the aglycon moiety showed a set of signals due to a furan
ring with 2,3-disubstituents at d 7.29 (d, JZ2.1 Hz) and
6.74 (d, JZ2.1 Hz), one olefinic proton at d 5.43 (br d, JZ
5.1 Hz), two carbinylic protons at d 4.03 (ddd, JZ11.7, 8.8,
4.4 Hz) and 3.63 (ddd, JZ11.5, 8.8, 5.4 Hz), and two
tertiary methyl groups at d 2.50 (s) and 1.03 (s) (Table 2).
The partial structures of the aglycon (C-1 to C-4, C-6 to C-9,
C-9 to C-12 and C-15 to C-16) were deduced from the
detailed analyses of the DQFCOSY spectrum of 1 (Fig. 2).
Interpretation of the HMBC spectral data led to the
connectivities of those three partial units and quaternary
Keywords: Glycosides; Cynanchum atratum; Asclepiadaceae.
*
Corresponding author. Tel.: C81 47 472 1391; fax: C81 47 472 1404;
e-mail: nikaido@phar.toho-u.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.04.015

5798
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
Figure 1. Structures of compounds 1–15.
carbons to construct the whole structure of the aglycon. The
HMBC correlations from dH 1.82 (H-9) to dC 45.0 (C-1), dH
5.43 (H-6) to dC 37.6 (C-4), as well as from dH 1.03 (H-19)
to dC 45.0 (C-1), 140.0 (C-5) and 48.3 (C-9) indicated the
connectivity of the A ring and B ring (Fig. 2). Further
evidence of the disubstituted furan ring was provided by the
HMBC correlations from dH 7.29 (H-15), 6.74 (H-16) and
2.50 (H-21) to dC 121.6 (C-17) and 157.9 (C-20),
respectively. Although the correlations from dH 2.30
(H-11a), 2.05 (H-11b) and 6.74 (H-16) to dC 196.0 (C-13)
was negative, while that of the second one [lmax 237 nm
(D3ZC19.13)] was positive, the chirality between the two
p-bromobenzoyloxy groups at C-2 and C-3 was a left-hand
screw, indicating 2R, 3R configurations. The same stereo-
chemical assignment of the aglycon was also confirmed by
the modified Mosher method.8 The methyl ester derivative
of 1 (17) was converted into its (R)- and (S)-di-MPTA ester
(22 and 23) by treatment with (S)- and (R)-a-methoxy-a-
(trifluoromethyl)-phenylacetyl chloride. As shown in
Scheme 1, significant Dd value (dSKdR, ppm) for the
protons near the derivatized chiral center C-2 was observed,
suggesting that C-2 was of R configuration. The type of the
sugar units and the sequence of the oligosaccharide chain of
1 were established by a combination of 2D NMR
experiments and chemical analysis. The DQFCOSY
spectrum showed that three of the sugar units belonged to
2,6-deoxy sugars (systems DS1, DS2 and DS3) because each
of them contained four methines, one methylene and a
terminal methyl group. Moreover, the location of the
methoxy groups was determined by the long-range HMBC
correlations (dH 3.55/dC 77.5 for DS1, dH 3.45/dC 74.6 for
DS2 and dH 3.47/dC 78.9 for DS3). In the spin systems DS1
and DS3, a large coupling constant between H-4 and H-5
(9.6 Hz) disclosed their trans-diaxial relationship, while a
small coupling constant of 2.8 Hz between H-3 and H-4
indicated a cis disposition of those protons. The above
evidence suggested the DS1 and DS3 sugar units were both
cymaropyranose and the b-glycosidic linkages were implied
by the J values (9.8, 1.6 Hz) of the anomeric protons. For the
remaining spin system, the signals assigned to H-3 at d 3.83
(ddd, JZ12.6, 4.6, 3.0 Hz) and H-4 at d 4.09 (br d, JZ
1.8 Hz), as well as NOESY correlation between H-3 and
H-5 presented the axial orientation of H-3 and H-5 and
the equatorial orientation of H-4. The data allowed the
identification of DS2 sugar as diginopyranose and the
a-configuration was inferred from the anomeric proton at d
5.18 (d, JZ3.5 Hz). The connection and the sequence of the
sugar chain at C-3 were elucidated by the results of the
HMBC experiment, which showed correlations between dH
3
though JCH were not observed, the correlations from dH
3.24 (H-12a) and 2.97 (H-12b) to dC 196.0 (C-13) though
2JCH and the correlation from dH 2.50 (H-21) to dC 196.0
(C-13) though 4JCH were obviously observed, indicating the
furan ring and C-12 was connected through a ketone carbon.
In addition, a carboxylic acid unit was attached at C-8 on the
basis of the correlation between dH 2.79 (H-8) and dC 178.9
(C-14). The relative stereochemistry of the aglycon was
elucidated by the 1H–1H coupling constants and the NOESY
spectrum. The coupling constant between H-2 and H-3
(8.8 Hz) was typical for trans-diaxial protons, indicating
that both oxygenated substituents were equatorial. Observed
1,3-diaxial NOE correlations for H-2/H-4b, H-2/H-19,
H-4b/H-19 and H-1a/H-3 (Fig. 3) further supported the
above result and revealed the chair conformation of the A
ring and the axial-orientation of H-19. The trans-diaxial
relationship of H-8 and H-9, namely, the b-orientation of
H-8 and a-orientation of H-9, was suggested by the splitting
pattern of H-8 (td, JZ11.5, 4.9 Hz) and the NOESY
correlations for H-8/H-19 and H-1a/H-9. Thus, the aglycon
was concluded as 15,20-epoxy-2a,3b-dihydroxy-
13,14:14,15-diseco-18-nor-pregna-5,15(16),17(20)-trien-
13-one-14-oic acid, which was obtained after acid hydrolysis
and named cynanogenin A (12) ([a]D K11.08). To
determine the absolute stereochemistry of 12, the methyl
ester derivative (16) was treated with p-bromobenzoyl
chloride to yield bis-p-bromobenzoate (19), to which the
exciton chirality method was applied (Scheme 1).7 As the
sign of the first Cotton effect [lmax 254 nm (D3ZK31.69)]

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
Table 1. 13C NMR data (500 MHz) for 1–11 in C5D5N
5799
Positions
1
2
3
4
5
6
7
8
9
10
44.8
70.0
85.4
11
44.8
1
2
3
4
5
6
7
8
45.0
70.0
85.2
37.6
140.0
120.9
30.5
43.0
48.3
39.5
23.5
45.0
70.0
85.2
37.6
45.0
70.0
85.2
37.6
140.0
120.9
30.6
43.0
48.3
39.5
23.6
45.0
69.6
85.2
37.3
139.4
121.0
28.3
38.3
55.1
40.1
23.0
110.5
145.3
173.5
141.3
109.7
115.7
44.8
70.0
85.4
37.6
139.6
120.9
28.6
40.3
52.7
39.6
30.7
44.9
70.0
85.3
37.6
44.8
70.0
85.4
37.6
139.7
120.9
28.6
40.3
52.7
39.6
30.7
44.9
70.0
85.4
37.6
139.7
120.9
28.6
40.3
52.7
39.6
30.7
44.8
70.0
85.4
37.6
139.8
120.8
28.5
40.3
53.1
39.5
30.1
70.0
85.4
37.6
139.8
120.8
28.5
40.3
53.1
39.5
30.1
23.8
114.4
175.4
67.8
75.6
56.2
143.9
19.0
118.6
24.8
37.6
140.0
120.9
30.6
139.7
120.9
28.6
139.8
120.8
28.5
40.3
53.1
39.5
30.1
23.9
114.4
175.4
67.8
75.6
56.2
143.9
19.0
118.6
24.8
43.0
48.3
39.5
23.6
40.3
52.8
39.6
30.7
9
10
11
12
13
14
15
16
17
18
19
20
21
42.3
42.4
42.4
20.8
20.8
20.8
20.8
23.8
196.0
178.9
140.7
110.7
121.6
196.0
179.0
140.7
110.7
121.6
196.0
179.0
140.7
110.7
121.6
119.0
175.5
67.2
82.1
92.5
144.7
19.0
119.7
20.7
119.0
175.5
67.2
119.0
175.5
67.2
82.1
92.4
144.7
19.0
119.8
20.7
119.0
175.5
67.2
82.1
92.5
144.7
19.0
119.8
20.7
114.4
175.4
67.8
75.6
56.2
143.9
19.0
118.6
24.8
82.1
92.5
144.7
19.0
119.8
20.7
19.3
157.9
14.2
19.3
158.0
14.2
19.3
157.9
14.2
20.5
150.0
13.6
b-D-cym I b-D-cym I b-D-cym
b-D-cym I b-D-cym I b-D-cym I b-D-cym
b-D-cym
b-D-cym I b-D-cym
b-D-cym I
1
2
3
4
97.4
35.2
77.5
82.1
69.6
18.4
57.4
97.5
35.2
77.5
82.1
69.6
18.3
57.4
97.8
37.0
77.9
82.9
69.4
18.2
59.0
97.5
35.2
77.5
82.1
69.6
18.4
57.4
97.5
35.2
77.5
82.1
69.6
18.4
57.4
97.5
35.2
77.5
82.1
69.6
18.4
57.4
97.9
37.0
77.9
82.9
69.4
18.2
59.0
97.9
37.1
77.9
83.0
69.4
18.2
59.0
97.5
35.2
77.5
82.1
69.6
18.4
57.4
97.9
37.0
77.9
82.9
69.4
18.2
59.0
97.5
35.2
77.5
82.1
69.6
18.4
57.4
5
6
3-OCH3
a-L-dgn
a-L-dgn
b-D-dgt
a-L-dgn
a-L-dgn
a-L-dgn
a-D-dgn
a-D-dgt
a-L-dgn
a-D-dgt
a-L-dgn
1
2
3
4
101.1
32.5
74.6
73.9
67.6
17.9
55.4
101.1
32.4
74.6
73.6
67.6
17.9
55.4
100.4
38.5
67.7
80.9
68.9
18.5
101.1
32.4
74.6
73.7
67.6
17.9
55.4
101.1
32.4
74.6
73.9
67.6
17.9
55.4
101.1
32.4
74.6
73.7
67.6
17.9
55.4
100.4
38.5
67.7
80.8
68.9
18.5
100.6
39.8
67.7
82.3
68.8
18.6
101.1
32.4
74.6
73.7
67.6
17.9
55.4
100.5
38.5
67.7
80.8
68.9
18.5
101.1
32.5
74.6
73.9
67.6
17.9
55.4
5
6
3-OCH3
b-D-cym
II
b-D-cym
II
a-L-cym
b-D-cym
II
b-D-cym
II
b-D-cym
II
a-L-cym
a-L-ole
b-D-cym
II
a-L-cym
b-D-cym
II
1
2
3
4
99.5
35.3
78.9
74.2
71.1
18.8
58.0
99.3
36.2
78.1
83.0
69.6
18.5
58.6
98.5
32.3
76.6
72.7
67.3
18.4
56.8
99.3
36.2
78.2
83.0
69.6
18.5
58.6
99.5
35.3
78.9
74.2
71.1
18.8
58.0
99.3
36.2
78.2
83.0
69.6
18.5
58.6
98.4
32.3
76.6
72.7
67.3
18.4
56.8
100.4
35.8
78.9
76.9
69.6
18.5
57.1
99.3
36.2
78.2
83.0
69.6
18.5
58.6
98.5
32.3
76.6
72.7
67.3
18.4
56.9
99.5
35.4
78.9
74.2
71.1
18.8
58.0
5
6
3-OCH3
b-D-glc
b-D-glc
b-D-glc
b-D-glc
1
2
3
4
5
6
106.5
75.4
78.4
71.9
78.4
63.0
106.5
75.5
78.4
71.9
78.4
63.0
106.5
75.5
78.4
71.9
78.4
63.0
106.5
75.5
78.4
71.9
78.4
63.0
cym, cymaropyranosyl; dgn, diginopyranosyl; dgt, digitoxopyranosyl; ole, oleandropyranosyl; glc, glucopyranosyl.
5.12 (cym II-H-1) and dC 73.9 (dgn-C-4), dH 5.18 (dgn-H-1)
and dC 82.1 (cym I-C-4), and dH 5.19 (cym I-H-1) and dC
85.2 (C-3). 2,6-Deoxy sugars in the glycosides from
Asclepiadaceae have been reported to be present as both
D- and L-enantiomers.9 To determine the absolute configur-
ations of 2,6-deoxy sugars in 1, it was hydrolyzed by 0.05 M
HCl (dioxane/H2O, 1:1) at 60 8C for 1.5 h, and analyzed by
HPLC with the detection by using the refractive index (RI)
and optical rotation (OR) detectors. The OR detection
exhibited a negative peak for the diginose and a positive one
for the cymaroses, suggesting L-form of the diginose and
D-form of the cymaroses. Based on the above results, the
structure of 1 was established as cynanogenin A 3-O-b-D-
cymaropyranosyl-(1/4)-a-L-diginopyranosyl-(1/4)-b-D-
cymaropyranoside.
Cynanoside B (2) was obtained as an amorphous powder. Its
molecular formula was determined to be C47H72O20 by
HRFABMS (m/z 979.4503 [MCNa]C, calcd for 979.4514).
Comparing the 1H and 13C NMR data of 2 with those of 1, 2

5800
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
Table 2. 1H NMR data (500 MHz) of 1–4 in C5D5N
Positions
1
2
3
4
1a
1b
2
1.41 (t, 12.4)
1.41 (t, 12.4)
2.61 (dd, 13.0, 4.4)
4.04a
3.63 (m)
2.53 (dd, 14.0, 5.5)
2.48 (m)
5.43 (br d, 5.3)
2.69 (m), 2.33 (m)
2.80 (td, 11.2, 4.8)
1.82a
2.30 (m), 2.05 (m)
3.24 (ddd, 16.8, 10.3, 4.9)
2.97 (ddd, 16.8, 10.1, 6.5)
7.29 (d, 2.1)
1.41 (t, 12.4)
1.21 (t, 12.4)
2.41a
2.61 (dd, 13.0, 4.4)
4.03 (ddd, 11.7, 8.8, 4.4)
3.63 (ddd, 11.5, 8.8, 5.4)
2.53 (dd, 13.9, 5.4)
2.48 (m)
5.43 (br d, 5.1)
2.69 (m), 2.32 (m)
2.79 (td, 11.5, 4.9)
1.82a
2.30 (m), 2.05 (m)
3.24 (ddd, 16.8, 10.3, 4.8)
2.97 (ddd, 16.8, 9.9, 6.4)
7.29 (d, 2.1)
6.74 (d, 2.1)
1.03 (s)
2.50 (s)
2.60 (dd, 13.3, 4.4)
4.01 (ddd, 11.7, 8.7, 4.4)
3.62a
2.51 (dd, 13.5, 6.0)
2.47 (m)
5.43 (br d, 5.1)
2.69 (m), 2.32 (m)
2.79 (td, 11.0, 4.9)
1.81 (ddd, 11.0, 7.3, 2.0)
2.29 (m), 2.03 (m)
3.24 (ddd, 16.8, 10.5, 5.1)
2.97 (ddd, 16.8, 10.0, 6.6)
7.29 (d, 2.1)
4.05a
3.58 (m)
3
4a
4b
6
7
8
2.49 (dd, 13.7, 5.7)
2.47 (m)
5.40 (br d, 5.3)
2.77 (m), 2.18 (m)
3.15 (td, 11.2, 4.8)
2.14 (m)
9
11
12a
12b
15
16
19
21
2.57 (m), 2.21 (m)
5.70 (dd, 8.4, 7.1)
7.46 (d, 2.1)
6.63 (d, 2.1)
1.05 (s)
6.74 (d, 2.1)
1.03 (s)
2.51 (s)
6.74 (d, 2.1)
1.02 (s)
2.51 (s)
2.41 (s)
b-D-cym I
b-D-cym I
b-D-cym
b-D-cym I
1
2a
2e
3
4
5
5.19 (dd, 9.8, 1.6)
1.82a
5.19 (dd, 9.6, 2.0)
1.82a
5.19 (dd, 9.6, 1.8)
1.87a
5.19 (dd, 9.6, 1.8)
1.82 (ddd, 13.7, 9.6, 2.3)
2.46a
3.92 (q like, 2.8)
3.45 (dd, 9.6, 2.8)
4.25 (dq, 9.6, 6.2)
1.32 (d, 6.2)
2.45a
2.48 (ddd, 13.5, 3.5, 2.0)
3.91 (q like, 2.8)
3.45 (dd, 9.6, 2.8)
4.25 (dq, 9.6, 6.2)
1.31 (d, 6.2)
2.33a
3.93 (q like, 2.8)
3.45a
4.26 (dq, 9.6, 6.2)
1.31 (d, 6.2)
3.55 (s)
4.08 (q like, 2.8)
3.48 (dd, 9.6, 2.8)
4.22 (dq, 9.6, 6.2)
1.30 (d, 6.2)
3.62 (s)
6
3-OCH3
3.54 (s)
3.55 (s)
a-L-dgn
a-L-dgn
b-D-dgt
a-L-dgn
1
2a
2e
3
4
5
5.18 (d, 3.5)
5.14 (br s)
2.33a
5.23 (dd, 9.6, 1.9)
1.91 (ddd, 13.5, 9.6, 3.0)
2.39a
5.14 (br s)
2.34 (td, 12.6, 3.9)
2.00 (dd, 12.6, 4.6)
3.83 (ddd, 12.6, 4.6, 3.0)
4.09 (br d, 1.8)
4.24a
2.34 (td, 12.5, 3.5)
2.00 (dd, 12.5, 4.5)
3.80 (ddd, 12.5, 4.5, 2.5)
4.05 (br d, 2.5)
4.23a
1.99 (dd, 12.4, 4.5)
3.80 (ddd, 12.4, 4.5, 2.9)
4.04 (br s)
4.46a
3.44 (dd, 9.6, 3.0)
4.14 (dq, 9.6, 6.2)
1.40 (d, 6.2)
4.24a
1.42 (d, 6.6)
3.41 (s)
6
3-OCH3
1.47 (d, 6.6)
3.45 (s)
1.42 (d, 6.6)
3.41 (s)
b-D-cym II
b-D-cym II
a-L-cym
b-D-cym II
1
2a
2e
3
4
5
5.12 (dd, 9.8, 1.6)
1.86 (ddd, 13.7, 9.8, 2.5)
2.47a
3.74 (q like, 2.8)
3.56a
4.12 (dq, 9.6, 6.2)
1.53 (d, 6.2)
3.47 (s)
5.15 (dd, 9.6, 2.0)
1.86 (ddd, 13.5, 9.6, 2.5)
2.37 (ddd, 13.5, 3.5, 2.0)
4.09 (q like, 2.8)
3.70 (dd, 9.8, 2.8)
4.28 (dq, 9.8, 6.2)
1.64 (d, 6.2)
5.09 (dd, 4.2, 2.5)
1.86a
5.15 (dd, 9.6, 1.6)
1.87 (ddd, 13.3, 9.6, 2.6)
2.39a
4.10 (q like, 2.8)
3.71 (dd, 9.6, 2.8)
4.28 (dq, 9.6, 6.2)
1.65 (d, 6.2)
2.40a
3.72 (q like, 3.3)
3.63a
4.48 (m)
1.44 (d, 6.4)
3.40 (s)
6
3-OCH3
3.52 (s)
3.52 (s)
b-D-glc
b-D-glc
1
2
3
4
5
4.93 (d, 7.8)
4.94 (d, 7.8)
4.01 (dd, 8.7. 7.8)
4.24 (t, 9.0)
4.20 (t, 9.0)
3.97 (ddd, 9.0, 5.3, 2.7)
4.40 (dd, 11.7, 5.3)
4.55 (dd, 11.7, 2.7)
4.02 (dd, 8.7. 7.8)
4.25 (t, 9.0)
4.21 (t, 9.0)
3.97 (ddd, 9.0, 5.3, 2.7)
4.40 (dd, 11.7, 5.3)
4.55 (dd, 11.7, 2.7)
6
cym, cymaropyranosyl; dgn, diginopyranosyl; dgt, digitoxopyranosyl; glc, glucopyranosyl.
a Overlapped signals.
had one more glucopyranose unit with the anomeric proton
signal resonating at d 4.93 (d, JZ7.8 Hz). Hydrolysis of 2
with naringinase gave the same result that only 1 and
D-glucose were detected from the hydrolyzate. The position
of the glucopyranosyl group in 2 was suggested to be
located at cym II-C-4 from the glycosidation shift observed
at cym II-C-3 (K0.8 ppm), cym II-C-4 (C8.8 ppm) and
cym II-C-5 (K1.5 ppm). This sugar linkage was also
supported by the HMBC correlation from dH 4.93 (glc-H-1)
to dC 83.0 (cym II-C-4). Thus, the structure of 2 was
established as cynanogenin A 3-O-b-D-glucopyranosyl-(1/
4)-b-D-cymaropyranosyl-(1/4)-a-L-diginopyranosyl-(1/
4)-b-D-cymaropyranoside.
Cynanoside C (3) was obtained as an amorphous powder. Its
molecular formula was determined to be C40H60O15 by
HRFABMS (m/z 803.3832 [MCNa]C, calcd for 803.3830).
The 1H and 13C NMR spectra indicated that 3 possessed the
same aglycon as that of 1, but differed in the sugar moiety.
The anomeric proton signals at d 5.23 (dd, JZ9.6, 1.9 Hz),
5.19 (dd, JZ9.6, 1.8 Hz) and 5.09 (dd, JZ4.2, 2.5 Hz) in
the 1H NMR spectrum revealed that 3 has three sugar units,

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5801
L-cymaroses occurred in the same molecule since OR of the
cymaroses was detected to be zero, namely, no peak of
the cymaroses was observed. The absolute configuration of
the terminal a-cymarose was elucidated by applying the
modified Mosher method after methylation of the carboxyl
group at C-14. Treatment of the methyl ester derivative (18)
with (S)- and (R)-MPTA chloride gave (R)- and (S)-MPTA
ester (24 and 25), respectively. The signals due to the
protons at a-cym-C-1, a-cym-C-2, a-cym-C-3 and a-cym-
OCH3 in the (S)-MPTA ester (25) were observed at lower
fields compared with those of (R)-MPTA ester (24) (Dd:
positive), while the signals due to protons at a-cym-C-5 and
a-cym-C-6 in 25 were observed at higher field compared
with those of 24. (Dd: negative) (Fig. 4). Consequently, the
absolute configuration of a-cym-C-4 was determined as S,
which indicated the terminal sugar is L-cymarose. Thus, the
structure of 3 was established as cynangenin A 3-O-a-L-
cymaropyranosyl-(1/4)-b-D-digitoxopyranosyl-(1/4)-b-
D-cymaropyranoside.
Figure 2. Key DQFCOSY and HMBC correlations of 1.
Figure 3. Observed NOESY correlations in the aglycon unit of 1.
Figure 4. Dd values (dSKdR) for the MPTA ester of 18 in ppm.
Cynanoside D (4) was obtained as an amorphous powder. Its
molecular formula was determined to be C47H70O19 by
HRFABMS (m/z 961.4396 [MCNa]C, calcd for 961.4409).
The NMR spectral comparison of 4 and 2 revealed that both
compounds had the same substitution pattern at C-3 and the
difference was only in the signals due to the aglycon,
including the absence of a ketone carbonyl carbon at d 196.0
and an alkyl carbon at d 42.4 with the appearance of two
olefinic carbons at d 145.3 (C-13) and 110.5 (C-12) in 4. The
assignment of each signal using 2D NMR data led to a
tricyclic system through an enol lactone linkage in the
structure of 4. The lactone ring formed between C-13 and
C-14 was supported by the HMBC correlations from dH 2.57
(H-11a), 2.21 (H-11b) and 2.41 (H-21) to dC 145.3 (C-13),
as well as dH 3.15 (H-8) to dC 173.5 (C-14) (Fig. 5). The
relative stereochemistry of the aglycon [named cynanogenin
B (13)] elucidated by NOESY correlations (Fig. 6) was
almost the same as that of 2. Treatment of 4 with 0.3%
NaOH/acetone (1:1) afforded 2, which was confirmed on the
basis of HPLC and TLC examination (Scheme 1). Thus, the
Scheme 1. The structures of S1 and S2 are shown as those in Figure 1.
(i) 0.05 M HCl (dioxane/H2O, 1:1), 60 8C, 1.5 h; (ii) CH2N2/Et2O, rt, 1 h;
(iii) p-bromobenzoyl chloride, N,N-dimethylamine pyridine, pyridine, rt,
24 h; (iv) (S) or (R)-MTPA-Cl, pyridine, rt, 4 h; (v) Naringinase, 0.1 M
acetate buffer, 40 8C, 24 h; (vi) 0.3% NaOH, acetone, rt, 24 h.
two of which were determined to be a-cymaropyranose and
b-cymaropyranose, and the other was elucidated as
b-digitoxopyranose by a combination of the DQFCOSY,
HMQC experiments and the 1H-1H coupling constants. The
connection and the sequence of the sugar chain was deduced
from observation of the HMBC correlations from dH 5.09
(a-cym-H-1) to dC 80.9 (dgt-C-4), dH 5.23 (dgt-H-1) to dC
82.9 (b-cym-C-4), and dH 5.19 (b-cym-H-1) to dC 85.2
(C-3). Acid hydroysis of 3, similarly as carried out on 1,
suggested that the digitoxose was D-form and both D- and
Figure 5. Key HMBC correlations of 4.

5802
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
structure of 4 was established as cynanogenin B 3-O-b-D-
glucopyranosyl-(1/4)-b-D-cymaropyranosyl-(1/4)-a-L-
diginopyranosyl-(1/4)-b-D-cymaropyranoside.
Cynanoside E (5) was obtained as an amorphous powder. Its
molecular formula was determined to be C42H64O16 by
HRFABMS (m/z 847.4111 [MCNa]C, calcd for 847.4092).
Of the 42 carbon signals observed in the 13C NMR spectrum
(Table 1), 21 were assigned to the aglycon with a pregnane
skeleton. A tertiary methyl signal at d 19.0, and two olefinic
Figure 6. Observed NOESY correlations in the aglycon unit of 4.
Table 3. 1H NMR data (500 MHz) for 5–8 in C5D5N
Positions
5
6
7
8
1a
1b
2
3
4
1.23 (t, 12.4)
2.47a
1.24 (t, 12.4)
2.47a
1.22 (t, 12.4)
2.48 (dd, 12.8, 4.6)
4.00a
3.56 (ddd, 11.2, 8.9, 5.7)
2.46a
1.22 (t, 12.4)
2.48a
4.02a
4.03a
4.00a
3.58a
3.59 (ddd, 11.6, 8.9, 5.8)
2.50a
3.57a
2.49a
2.47a
6
7
8
9
11
12
15a
15b
16
17
18
19
21
5.42 (br d, 5.3)
2.68 (m), 2.16 (m)
2.54 (td, 11.0, 4.7)
1.38 (t, 11.0)
2.23 (m), 1.56 (m)
2.47 (m), 1.85 (m)
4.32 (dd, 9.4, 7.3)
4.01 (t, 9.1)
5.41 (br d, 5.3)
2.70 (m), 2.16 (m)
2.54 (td, 10.9, 4.9)
1.38a
2.23 (m), 1.57 (m)
2.47 (m), 1.84 (m)
4.32 (dd, 9.4, 7.4)
4.01 (t, 9.0)
5.42 (br d, 5.1)
2.69 (m), 2.16 (m)
2.53 (td, 11.1, 4.8)
1.38a
2.22 (m), 1.55 (m)
2.48 (m), 1.85 (m)
4.32 (dd, 9.2, 7.4)
4.01 (t, 9.1)
5.42 (br d, 5.3)
2.69 (m), 2.16 (m)
2.54a
1.38 (t, 10.7)
2.22 (m), 1.57 (m)
2.47 (m), 1.83 (m)
4.32 (dd, 9.3, 7.4)
4.01 (t, 9.2)
6.00 (dd, 8.5, 7.4)
6.00 (dd, 8.7, 7.3)
6.01 (dd, 8.8, 7.4)
6.00 (dd, 8.9, 7.4)
6.67 (s)
0.94 (s)
1.76 (s)
6.67 (s)
0.94 (s)
1.77 (s)
6.67 (s)
0.93 (s)
1.76 (s)
6.67 (s)
0.92 (s)
1.77 (s)
b-D-cym I
b-D-cym I
b-D-cym
b-D-cym
1
2a
2e
3
4
5
5.18 (dd, 9.6, 1.8)
1.82a
5.18 (dd, 9.8, 1.8)
1.81 (ddd, 13.7, 9.8, 2.4)
2.47a
3.91 (q like, 2.8)
3.44 (dd, 9.6, 2.8)
4.26 (dq, 9.6, 6.2)
1.31 (d, 6.2)
5.18 (dd, 9.8, 2.0)
1.87a
5.20 (dd, 9.8, 2.0)
1.91 (ddd, 13.5, 9.8, 2.5)
2.36 (ddd, 13.5, 3.5, 2.0)
4.11 (q like, 2.8)
3.49 (dd, 9.6, 2.8)
4.23 (dq, 9.6, 6.3)
1.30 (d, 6.3)
2.44a
2.33 (ddd, 13.3, 3.2, 2.0)
4.07 (q like, 2.8)
3.47 (dd, 9.8, 2.8)
4.21 (dq, 9.8, 6.2)
1.29 (d, 6.2)
3.92 (q like, 2.8)
3.45a
4.26 (dq, 9.7, 6.2)
1.32 (d, 6.2)
3.55 (s)
6
3-OCH3
3.54 (s)
3.62 (s)
3.65 (s)
a-L-dgn
a-L-dgn
b-D-dgt
b-D-dgt
1
2a
2e
3
4
5
5.16 (d, 4.3)
5.13 (br d, 2.0)
2.33 (td, 12.4, 3.7)
1.99 (dd, 12.4, 4.4)
3.80 (ddd, 12.4, 4.4, 2.9)
4.04 (br d, 2.1)
4.23a
5.22 (dd, 9.6, 1.7)
1.91 (ddd, 13.8, 9.6, 2.7)
2.39 (ddd, 13.8, 3.4, 1.7)
4.46a
3.44 (dd, 9.6, 3.0)
4.13 (dq, 9.6, 6.2)
1.39 (d, 6.2)
5.36 (dd, 9.6, 1.6)
1.97 (ddd, 13.3, 9.6, 2.5)
2.41a
4.60 (q like, 2.8)
3.53a
2.34 (td, 12.4, 3.8)
1.99 (dd, 12.4, 4.4)
3.82 (ddd, 12.4, 4.4, 2.9)
4.08 (br d, 1.9)
4.23a
4.40 (dq, 9.8, 6.4)
1.45 (d, 6.4)
6
3-OCH3
1.46 (d, 6.4)
3.44 (s)
1.41 (d, 6.4)
3.41 (s)
b-D-cym II
b-D-cym II
a-L-cym
a-L-ole
1
2a
2e
3
4
5
5.12 (dd, 9.8, 1.9)
1.85 (ddd, 13.4, 9.8, 2.5)
2.46a
3.73 (q like, 3.0)
3.56a
4.11 (dq, 9.6, 6.2)
1.53 (d, 6.2)
3.47 (s)
5.14 (dd, 9.6, 1.8)
1.87 (ddd, 13.4, 9.6, 2.5)
2.38 (ddd, 13.4, 3.4, 1.8)
4.10 (q like, 2.8)
3.71 (dd, 9.6, 2.8)
4.28 (dq, 9.6, 6.2)
1.65 (d, 6.2)
5.09 (dd, 4.1, 2.3)
1.86a
5.26 (d, 3.7)
1.75 (m)
2.40a
2.49a
3.72 (q like,3.7)
3.62a
4.48 (m)
1.44 (d, 6.4)
3.40 (s)
3.84 (ddd, 11.5, 9.0, 4.8)
3.54 (t, 9.0)
4.41 (dq, 9.6, 6.2)
1.52 (d, 6.2)
3.35 (s)
6
3-OCH3
3.52 (s)
b-D-glc
1
2
3
4
5
4.94 (d, 7.8)
4.02 (dd, 8.8, 7.8)
4.24 (t, 8.9)
4.20 (t, 8.9)
3.97 (ddd, 8.9, 5.3, 2.5)
4.40 (dd, 11.7, 5.3)
4.56 (dd, 11.7, 2.5)
6
cym, cymaropyranosyl; dgn, diginopyranosyl; dgt, digitoxopyranosyl; ole, oleandropyranosyl; glc, glucopyranosyl.
a Overlapped signals.

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5803
carbons at d 139.6 and 120.9, coupled with the information
from the 1H NMR data (Table 3) (an angular methyl proton
singlet at d 0.94 and a broad doublet olefinic proton signal at
Cynanoside G (7) was obtained as an amorphous powder. Its
molecular formula was determined to be C41H62O16 by
HRFABMS (m/z 833.3963 [MCNa]C, calcd for 833.3936).
On acid hydrolysis, 7 afforded cymarose (a mixture of D-
and L-form in the ratio 1:1), D-digitoxose and 14. From its
1H and 13C NMR (Tables 1 and 3), it was evident that 7
possessed the same sugar moiety at C-3 as that of 3. Thus,
the structure of 7 was established as hancopregnane 3-O-a-
L-cymaropyranosyl-(1/4)-b-D-digitoxopyranosyl-(1/4)-
b-D-cymaropyranoside.
1
d 5.42), indicated the aglycon is a D5-pregnane. The H
NMR spectrum of the aglycon moiety also showed signals
for the methyl at d 1.76 (s), one olefinic proton signal at d
6.67 (s) connected with the trisubstituted double bond, three
oxygen-substituted methine protons at d 6.00 (dd, JZ8.7,
7.3 Hz), 4.02 and 3.58, and two oxygen-substituted
methylene protons at d 4.32 (dd, JZ9.4, 7.3 Hz) and 4.01
(t, JZ9.1 Hz), which exhibited the characteristic feature of
13,14:14,15-diseco-pregnane-type steroidal glycoside.10
Acid hydrolysis of 5 allowed the aglycon to be identified
as hancopregnane (14) by comparison of their physical and
spectroscopic data.11 Although the gross structure and the
relative stereochemistry of 14 have been established by
X-ray analysis, the absolute stereochemistry remains to be
defined. The absolute configuration of 14 was elucidated by
the exciton chirality method. In the CD spectrum of
p-bromobenzoate derivative of 14 (20), a split CD curve
having a negative Cotton effect at 254 nm (D3ZK39.04)
and a positive one at 238 nm (D3ZC32.90) was observed,
indicating negative chirality between the two chromo-
phores. Therefore, C-2 and C-3 were assigned as both R
configurations. The assignment was also confirmed by the
application of the modified Mosher method for 5 (Fig. 7).
The identities of the monosaccharides and the oligo-
saccharide sequence were determined by extensive analysis
of 1D and 2D NMR spectra, as well as the results of acid
hydrolysis with the same method as 1. Since, the
glycosidation shifts were observed at C-2 (K2.4 ppm),
C-3 (C8.7 ppm) and C-4 (K2.6 ppm), the sugar moiety was
linked to the C-3 hydroxyl group of the aglycon. On the basis
of all the above evidence, the structure of 5 was established as
hancopregnane 3-O-b-D-cymaropyranosyl-(1/4)-a-L-
diginopyranosyl-(1/4)-b-D-cymaropyranoside.
Cynanoside H (8) was obtained as an amorphous powder.
The HRFABMS of 8 suggested the molecular formula of
C41H62O16 (m/z 833.3953 [MCNa]C, calcd for 833.3936),
which is the same as that of 7. Comparison of the NMR
spectra of 7 and 8 indicated that 8 differed from 7 only by
the replacement of the terminal cymaropyranose by an
oleandropyranose, which was deduced from the large
coupling constant of H-3 and H-4 (9.0 Hz). The aglycon,
D-cymarose and D-digitoxose were identified by comparison
with authentic samples after acid hydrolysis, while the
absolute configuration of oleandrose was determined by the
modified Mosher method due to the lack of an authentic
sugar of oleandrose. The (R)- and (S)-MPTA ester of 8 (28
and 29) were synthesized and the values of the chemical
shift differences (DdZdSKdR, ppm) were calculated. As
shown in Figure 8, the Dd values for protons at ole-C-2, ole-
C-3 and ole-OCH3 are positive, while negative Dd values
were observed for protons of ole-C-5 and ole-C-6,
suggesting S configuration of ole-C-4, namely, L-form of
oleandrose. Thus, the structure of 8 was established as
hancopregnane 3-O-a-L-oleandropyranosyl-(1/4)-b-D-
digitoxopyranosyl-(1/4)-b-D-cymaropyranoside.
Figure 8. Dd values (dSKdR) for the MPTA ester of 8 in ppm.
Cynanoside I (9) was obtained as an amorphous powder. Its
molecular formula was determined to be C48H74O20 by
HRFABMS (m/z 993.4698 [MCNa]C, calcd for 993.4671).
The NMR data of 9 (Tables 1 and 4) were superimposable to
those of 6, except for the signals due to the furofuran
moiety. The upfield shift of the ring protons and carbons
were explainable by the absence of C-17-OH. Enzymatic
hydrolysis of 9 gave D-glucose and a known compound (11),
which was further hydrolyzed with 0.05 M HCl to give
D-cymarose, L-diginose and glaucogenin A (15).10b Since,
there was no study on the absolute stereochemistry of 15, we
reported here the determination of the absolute stereo-
chemistry of 15. The CD spectrum of the p-bromobenzoate
derivative of 15 (21) exhibited a negative Cotton effect at
253 nm (D3ZK35.24) and a positive one at 238 nm
(D3ZC29.76), indicating 2R and 3R configurations.
Based on the above results, the structure of 9 was
established as glaucogenin A 3-O-b-D-glucopyranosyl-
Figure 7. Dd values (dSKdR) for the MPTA ester of 5 in ppm.
Cynanoside F (6) was obtained as an amorphous powder. Its
molecular formula was determined to be C48H74O21 by
HRFABMS (m/z 1009.4646 [MCNa]C, calcd for
1009.4626). The overall structure assignment was accom-
plished by a combination of 1D and 2D NMR spectra. From
1
its H and 13C NMR data, it was apparent that 6 possessed
the same aglycon as that of 5 and the same sugar structures
as those of 2. Enzymatic hydrolysis of 6 gave D-glucose and
a deglucosyl derivative, which was identical as 5. Thus, the
structure of 6 was established as hancopregnane 3-O-b-D-
glucopyranosyl-(1/4)-b-D-cymaropyranosyl-(1/4)-a-L-
diginopyranosyl-(1/4)-b-D-cymaropyranoside.

5804
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
Table 4. 1H NMR data (500 MHz) for 9–11 in C5D5N
Positions
9
10
11
1a
1b
2
3
4
1.26 (t, 12.4)
2.49a
1.25 (t, 12.4)
2.49a
4.03 (ddd, 11.7, 8.8, 4.5)
1.24 (t, 12.4)
2.49a
4.01 (ddd, 11.5, 8.7, 4.6)
3.60 (ddd, 11.4, 8.7, 5.8)
2.49a
4.04a
3.62 (ddd, 11.7, 9.0, 5.6)
2.49a
3.60a
2.49a
6
7
8
9
5.41 (br d, 5.5)
2.67 (m), 2.17 (m)
2.53a
5.43 (br d, 4.0)
2.67 (m), 2.16 (m)
2.51a
5.40 (br d, 5.5)
2.65 (m), 2.15 (m)
2.52a
1.33a
1.32a
1.32a
11
12
15a
15b
16
17
18
19
21
2.12 (m), 1.39 (m)
2.56 (m), 1.36 (m)
4.25 (dd, 8.7, 7.0)
3.95 (t, 9.2)
5.45 (dt, 9.4, 7.6)
3.56a
6.49 (s)
0.92 (s)
1.55 (s)
2.11 (m), 1.41 (m)
2.55 (m), 1.36 (m)
4.25 (dd, 8.6, 7.0)
3.96 (dd, 9.6, 8.6)
5.45 (m)
3.55 (d, 8.0)
6.49 (s)
0.91 (s)
2.12 (m), 1.41 (m)
2.56 (m), 1.37 (m)
4.24a
3.94 (t, 9.6)
5.43 (m)
3.54a
6.47 (s)
0.91 (s)
1.54 (s)
1.55 (s)
b-D-cym I
b-D-cym
b-D-cym I
1
2a
2e
3
4
5
5.20 (dd, 9.8, 2.0)
1.82 (ddd, 13.7, 9.8, 2.2)
2.47a
3.91 (q like, 2.8)
3.45 (dd, 9.6, 2.8)
4.27a
5.21 (dd, 9.7, 2.0)
1.88a
5.18 (dd, 9.7, 1.6)
1.82 (ddd, 13.6 9.7, 2.1)
2.35a
2.44a
4.09 (q like, 2.8)
3.48 (dd, 9.6, 2.8)
4.23 (dq, 9.6, 6.2)
1.30 (d, 6.2)
3.62 (s)
3.92a
3.44a
4.24a
6
3-OCH3
1.32 (d, 6.2)
3.55 (s)
1.32 (d, 6.2)
3.54 (s)
a-L-dgn
b-D-dgt
a-L-dgn
1
2a
2e
3
4
5
5.14 (br s)
5.24 (dd, 9.7, 1.9)
1.92 (ddd, 13.9, 9.7, 2.8)
2.39a
5.15 (d, 3.2)
2.34 (td, 12.4, 3.7)
1.99 (dd, 12.4, 4.4)
3.80 (ddd, 12.4, 4.4, 3.0)
4.05 (br s)
2.34 (td, 12.6, 3.7)
1.99 (dd, 12.6, 4.4)
3.81 (ddd, 12.6, 4.4, 3.0)
4.07 (br s)
4.48a
3.45 (dd, 9.6, 3.0)
4.15 (dq, 9.6, 6.3)
1.40 (d, 6.3)
4.21a
1.42 (d, 6.4)
3.41 (s)
4.24a
1.45 (d, 6.4)
6
3-OCH3
3.43 (s)
b-D-cym II
a-L-cym
b-D-cym II
1
2a
2e
3
4
5
5.14 (dd, 9.6, 2.0)
1.87 (ddd, 13.6, 9.6, 2.2)
2.38 (ddd, 13.6, 3.6, 2.0)
4.10 (q like, 2.8)
3.71 (dd, 9.6, 2.8)
4.28 (dq, 9.6, 6.4)
1.65 (d, 6.4)
5.11 (dd, 4.1, 2.5)
1.86a
5.12 (dd, 9.5, 1.5)
1.84 (ddd, 13.5, 9.5, 2.5)
2.46a
3.72 (q like, 2.8)
3.54a
4.10 (dq, 9.6, 6.2)
1.53 (d, 6.2)
3.46 (s)
2.41a
3.74 (q like, 3.5)
3.62a
4.51 (m)
1.44 (d, 6.4)
3.41 (s)
6
3-OCH3
3.52 (s)
b-D-glc
1
2
3
4
5
4.93 (d, 7.5)
4.01 (dd, 8.7, 7.5)
4.22 (t, 8.7)
4.20 (t, 8.7)
3.97 (ddd, 8.7, 5.2, 2.5)
4.40 (dd, 11.7, 5.2)
4.56 (dd, 11.7, 2.5)
6
cym, cymaropyranosyl; dgn, diginopyranosyl; dgt, digitoxopyranosyl; glc, glucopyranosyl.
a Overlapped signals.
(1/4)-b-D-cymaropyranosyl-(1/4)-a-L-diginopyranosyl-
(1/4)-b-D-cymaropyranoside.
glaucogenin A (15) as that of 9. On acid hydroysis, 10
gave cymarose (a mixture of D- and L-form in the ratio
1:1), D-digitoxose and 15. Although the specific rotation
and the NMR data of 10 were superimposable to
those of a previously reported compound, glaucoside
C,10b the inner cymarose was determined as b-D-
cymarose instead of b-L-cymarose. Thus, the structure
of 10 was established as glaucogenin A 3-O-a-L-
cymaropyranosyl-(1/4)-b-D-digitoxopyranosyl-(1/4)-
b-D-cymaropyranoside.
Cynanoside J (10) was obtained as an amorphous
powder. Its molecular formula was determined to be
C41H62O15 by HRFABMS (m/z 817.4023 [MCNa]C,
calcd for 817.3986). Comparison of the 1H and 13C
NMR spectra of 10 with those of 3 and 9, suggested
that the trisaccharide structure attached to C-3 of the
aglycon was identical to that of 3, while the aglycon was

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5805
The isolated glycosides 1–11 were evaluated for their
cytotoxic activitiy against HL-60 human promyelocytic
leukemia cells.12 7, 10 and 11 showed moderate cytotoxic
activity against HL-60 cells with IC50 values of 52, 32 and
24 mM, respectively, compared with that of cisplatine used
as a positive control (IC50 0.41 mM). 1–6, 8 and 9 were
inactive at 100 mM.
s, –OCH3), 3.23 (1H, m, H-4), 2.35 (1H, ddd, JZ14.1, 3.5,
2.2 Hz, H-2e), 1.52 (1H, ddd, JZ14.1, 9.6, 2.5 Hz, H-2a),
1.30 (3H, d, JZ6.4 Hz, H-6); for a-cymarofuranose, 5.48
(1H, d, JZ4.6 Hz, H-1), 3.37 (3H, s, –OCH3), 1.24 (3H, d,
JZ6.6 Hz, H-6); for b-cymarofuranose, 5.63 (1H, dd, JZ
5.7, 2.2 Hz, H-1), 3.30 (3H, s, –OCH3), 1.23 (3H, d, JZ
6.4 Hz, H-6). 13C NMR (125 MHz, CDCl3): for a-cymar-
cymaropyranose, 91.6 (C-1), 78.3 (C-3), 72.4 (C-4), 64.4
(C-5), 58.2 (–OCH3), 31.9 (C-2), 18.1 (C-6); for b-cymar-
cymaropyranose, 91.9 (C-1), 77.1 (C-3), 72.3 (C-4), 71.0
(C-5), 57.1 (–OCH3), 34.7 (C-2), 18.2 (C-6); for
a-cymarofuranose, 99.1 (C-1), 87.5 (C-3), 80.4 (C-4),
67.4 (C-5), 56.9 (–OCH3), 39.3 (C-2), 18.7 (C-6); for
b-cymarofuranose, 98.7 (C-1), 88.9 (C-3), 79.1 (C-4), 67.7
(C-5), 56.8 (–OCH3), 40.9 (C-2), 18.1 (C-6).
3. Experimental
3.1. General
The UV spectra were obtained with a SHIMADZU
BIOSPEC-MINI spectrophotometer, whereas the IR spectra
were measured with a JASCO FT/IR-300E (by a KBr disk
method) spectrometer. Optical rotations were measured
with a JASCO DIP-370 digital polarimeter in a 0.5 dm
length cell while the CD spectra were recorded on a JASCO
J-720W spectropolarimeter. The FABMS and HRFABMS
were taken on a JEOL JMS-700 Mstation spectrometer. The
3.2.2. L-Diginose (31). [a]D K16.18 (c 0.50, H2O, 24 8C,
24 h after dissolution). 1H NMR [(500 MHz, CDCl3);
w2:1:1:1 mixture of a-diginopyranose, b-diginopyranose,
a-diginofuranose and b-diginofuranose]: for a-digino-
pyranose, 5.42 (1H, br d, JZ2.8 Hz, H-1), 4.13 (q, JZ
6.6 Hz, H-5), 3.81 (1H, br d, JZ1.5 Hz, H-4), 3.71 (1H,
ddd, JZ11.8, 5.2, 3.1 Hz, H-3), 3.41 (1H, s, –OCH3), 1.94
(1H, dd, JZ13.0, 5.2 Hz, H-2e), 1.85 (1H, ddd, JZ13.0,
11.8, 3.5 Hz, H-2a), 1.31 (1H, d, JZ6.6 Hz, H-6); for
b-diginopyranose: 4.73 (1H, br d, JZ8.6 Hz, H-1; for
a-diginofuranose, 5.64 (1H, dd, JZ5.5, 2.1 Hz, H-1); for
b-diginofuranose, 5.49 (1H, m, H-1). 13C NMR (125 MHz,
CDCl3): for a-diginopyranose: 92.3 (C-1), 74.2 (C-3), 67.8
(C-4), 65.6 (C-5), 55.6 (–OCH3), 29.4 (C-2), 16.9 (C-6).
1
ESIMS was taken on an LCQ mass analyzer. The H and
13C NMR spectra were measured with a JEOL ECP-500
spectrometer with TMS as the internal reference, and
chemical shifts are expressed in d (ppm). HPLC separations
were carried out with a JASCO PU-2080 HPLC system,
equipped with a Shodex RI-101 Differential Refractometer
detector and a Senshu Pak RP-C18 column (150!20 mm
i.d.). For Lobar Normal-phase silica gel chromatography, a
JASCO PU-980 HPLC system and a Lichroprep Si 60
column (40–63 mM, Merck) were used. Reversed-phase
column chromatography (CC) was accomplished with RP-
C18 silica gel (100–200 mesh, Chromatorex DM1020T
ODS, Fuji Silysia Chemical Ltd). Silica gel CC was carried
out using Silica Gel 60N (Kanto Chemical Co., Inc). TLC
was conducted in Kieselgel 60 F254 plates (E. Merck).
3.2.3. D-Digitoxose (32). [a]D C50.28 (c 0.27, H2O, 24 8C,
24 h after dissolution). 1H NMR [(500 MHz, CDCl3); w7:1
mixture of a-digitoxopyranose and b-digitoxopyranose plus
minor amounts of furanose epimers]: for a-digitoxo-
pyranose, 5.25 (1H, d, JZ3.4 Hz, H-1), 4.08 (1H, d like,
JZ2.9 Hz, H-3), 4.04 (1H, dq, JZ9.7, 6.2 Hz, H-5), 3.25
(1H, dd, JZ9.7, 3.1 Hz, H-4), 2.23 (1H, ddd, JZ14.7, 3.5,
1.3 Hz, H-2e), 1.89 (1H, ddd, JZ14.7, 3.5, 3.0 Hz, H-2a),
1.33 (3H, d, JZ6.2 Hz, H-6); for b-digitoxopyranose, 5.18
(1H, dd, JZ9.8, 2.1 Hz, H-1), 4.14 (1H, d like, JZ3.2 Hz,
H-3), 3.80 (1H, dq, JZ9.6, 6.2 Hz, H-5), 3.24 (1H, dd, JZ
9.6, 3.2 Hz, H-4), 2.21 (1H, ddd, JZ14.0, 3.5, 2.2 Hz,
H-2e), 1.68 (1H, ddd, JZ14.0, 9.8, 3.0 Hz, H-2a), 1.32 (3H,
d, JZ6.2 Hz, H-6). 13C NMR (125 MHz, CDCl3): for
a-digitoxopyranose, 92.0 (C-1), 74.6, 73.9, 67.6 (C-3, C-4
and C-5), 32.5 (C-2), 17.9 (C-6).
3.2. Isolation of D-cymarose, L-diginose and D-digtoxose
Atratoside B6c (100 mg) was heated at 60 8C for 1.5 h with
0.05 M HCl (dioxane/H2O, 1:1, 2 mL). After dioxane was
removed, the solution was extracted with EtOAc (2 mL!3)
to remove the aglycon. The aqueous layer was neutralized
by passing through an ion-exchange resin (Amberlite MB-3,
Organo, Tokyo, Japan) column and concentrated under
reduced pressure to give the sugar fraction, which was
chromatographed on a silica gel column with CHCl3/MeOH
(94:6) to give D-cymarose (30, 8 mg) and L-diginose (31,
10 mg). Using a similar procedure, D-digitoxose (32, 10 mg)
was isolated from cynascyroside C (90 mg)13 by silica gel
column with CHCl3/MeOH (9:1).
3.3. Plant material
Plant material used in this research was collected from
Hebei province in China and identified as C. atratum Bge.
by Prof. Qishi Sun (Shenyang Pharmaceutical University).
3.2.1. D-Cymarose (30). [a]D C18.78 (c 0.24, H2O, 24 8C,
24 h after dissolution). 1H NMR [(500 MHz, CDCl3);
w18:12:9:7 mixture of a-cymaropyranose, b-cymaro-
pyranose, a-cymarofuranose and b-cymarofuranose]: for
a-cymaropyranose, 5.11 (1H, br d, JZ4.5 Hz, H-1), 3.96
(1H, dq, JZ9.9, 6.4 Hz, H-5), 3.74 (1H, q like, JZ2.7 Hz,
H-3), 3.54 (3H, s, –OCH3), 3.24 (1H, m, H-4), 2.32 (1H,
ddd, JZ14.8, 3.5, 1.3 Hz, H-2e), 1.77 (1H, ddd, JZ14.8,
3.7, 2.6 Hz, H-2a), 1.33 (3H, d, JZ6.4 Hz, H-6); for
b-cymaropyranose, 5.02 (1H, dd, JZ9.6, 1.8 Hz, H-1), 3.99
(1H, dq, JZ9.1, 6.4 Hz, H-5), 3.65 (1H, m, H-3), 3.44 (3H,
3.4. Extraction and isolation
The roots of C. atratum (1.8 kg) were extracted with MeOH
(3 L!5). Evaporation of the solvent under reduced pressure
from the combined extract gave the MeOH extract (285 g).
The extract was then partitioned between EtOAc and H2O.
The EtOAc layer (162 g) was subjected to silica gel CC with
a gradient of CHCl3/MeOH to give four fractions (1–4).
Fraction 2 (82.9 g) was loaded on a Diaion HP-20 column

5806
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
and eluted with 50%, 70%, 90% and 100% MeOH. The 90%
MeOH fraction (25.6 g) was further separated by chroma-
tography on a silica gel column using CHCl3/MeOH (99:1,
97:3, 95:5 and 9:1) to give five fractions (A–E). Fraction B
(6.17 g) was subjected to Sephadex LH-20 CC with MeOH
to give three fractions (B1–B3). Fraction B2 (1.72 g) was
purified by HPLC (60% CH3CN) to give 11 (70 mg).
Fraction C (3.00 g) was separated by Sephadex LH-20 CC
with MeOH to give three fractions (C1–C3). The major
fraction C2 (1.77 g) was further purified by HPLC with 45%
CH3CN to give 5 (70 mg) and with 60% CH3CN to give 10
(3 mg). Fraction D (7.04 g) was chromatographed on a
Sephadex LH-20 column with MeOH to give three
fractions (D1–D3). Fraction D2 (4.67 g) was fractionated
by HPLC (50% CH3CN) and Lobar Normal-phase silica gel
CC [n-hexane/CHCl3/EtOH (45:50:5)] to give 1 (98 mg), 3
(123 mg), 7 (45 mg) and 8 (8 mg). Further separation of
fraction 3 (19.1 g) was achieved by ODS CC with aqueous
MeOH with the ratios of 50%, 80% and 100%. The fraction
eluted with 80% MeOH (7.90 g) was purified by HPLC
(45% CH3CN) to give 2 (64 mg), 4 (10 mg), 6 (13 mg) and 9
(95 mg).
3.4.6. Cynanoside F (6). Amorphous powder, [a]D K25.38
(c 1.06, CHCl3, 24 8C). IR (KBr) nmax: 3450, 2925, 1738,
1687, 1067 cmK1. FABMS (positive) m/z: 1009 [MCNa]C.
HRFABMS (positive): observed 1009.4646, calcd for
C48H74O21Na [MCNa]C, 1009.4626. 1H NMR
(500 MHz, C5D5N) and 13C NMR (125 MHz, C5D5N): see
Tables 1 and 3.
3.4.7. Cynanoside G (7). Amorphous powder, [a]D K40.58
(c 1.02, CHCl3, 24 8C). IR (KBr) n max: 3446, 2932, 1737,
1687, 1090 cmK1. FABMS (positive) m/z: 833 [MCNa]C.
HRFABMS (positive): observed 833.3963, calcd for
1
C41H62O16Na [MCNa]C, 833.3936. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 3.
3.4.8. Cynanoside H (8). Amorphous powder, [a]D K7.738
(c 0.73, CHCl3, 24 8C). IR (KBr) nmax: 3445, 2930, 1721,
1687, 1055 cmK1. FABMS (positive) m/z: 833 [MCNa]C.
HRFABMS (positive): observed 833.3953, calcd for
1
C41H62O16Na [MCNa]C, 833.3936. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 3.
3.4.1. Cynanoside A (1). Amorphous powder, [a]D K26.58
(c 0.93, CHCl3, 24 8C). IR (KBr) nmax: 3423, 2926, 1737,
1687, 1065 cmK1. FABMS (positive) m/z: 817 [MCNa]C.
HRFABMS (positive): observed 817.4023, calcd for
3.4.9. Cynanoside I (9). Amorphous powder, [a]D K10.58
(c 1.10, CHCl3, 24 8C). IR (KBr) nmax: 3423, 2929, 1737,
1687, 1070 cmK1. FABMS (positive) m/z: 993 [MCNa]C.
HRFABMS (positive): observed 993.4698, calcd for
1
C41H62O15Na [MCNa]C, 817.3986. H NMR (500 MHz,
1
C48H74O20Na [MCNa]C, 993.4671. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 2.
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 4.
3.4.2. Cynanoside B (2). Amorphous powder, [a]D K25.58
(c 1.08, MeOH, 24 8C). IR (KBr) nmax: 3423, 2928, 1733,
1686, 1067 cmK1. FABMS (positive) m/z: 979 [MCNa]C.
HRFABMS (positive): observed 979.4503, calcd for
3.4.10. Cynanoside J (10). Amorphous powder, [a]D
K11.58 (c 0.25, CHCl3, 24 8C). IR (KBr) nmax: 3425, 2926,
1739, 1653, 1058 cmK1. FABMS (positive) m/z: 817 [MC
Na]C. HRFABMS (positive): observed 817.3977, calcd for
1
C47H72O20Na [MCNa]C, 979.4514. H NMR (500 MHz,
1
C41H62O15Na [MCNa]C, 817.3986. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 2.
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 4.
3.4.3. Cynanoside C (3). Amorphous powder, [a]D K44.38
(c 0.91, CHCl3, 24 8C). IR (KBr) nmax: 3432, 2930, 1721,
1656, 1058 cmK1. FABMS (positive) m/z: 803 [MCNa]C.
HRFABMS (positive): observed 803.3832, calcd for
3.5. Acid hydrolysis of compounds 1, 5 and 11
Each of 1 (10.9 mg), 5 (9.2 mg) and 11 (10.0 mg) was
heated at 60 8C for 1.5 h with 0.05 M HCl (dioxane/H2O,
1:1, 1 mL). After dioxane was removed, the solution was
extracted with EtOAc (2 mL!3) and further purified by
HPLC to give 12 (2.2 mg) (with 50% MeOH as eluate
solvent), 14 (2.9 mg) (55% MeOH) and 15 (2.6 mg) (60%
MeOH), respectively. The aqueous layer was neutralized by
passing through an ion-exchange resin (Amberlite MB-3,
Organo, Tokyo, Japan) column and concentrated under
reduced pressure to give the sugar fraction.
1
C40H60O15Na [MCNa]C, 803.3830. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 2.
3.4.4. Cynanside D (4). Amorphous powder, [a]D K19.88
(c 1.00, MeOH, 24 8C). IR (KBr) nmax: 3423, 2926, 1717,
1687, 1068 cmK1. FABMS (positive) m/z: 961 [MCNa]C.
HRFABMS (positive): observed 961.4396, calcd for
1
C47H70O19Na [MCNa]C, 961.4409. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 2.
3.5.1. Compound 12. [a]D K11.08 (c 0.22, CHCl3, 24 8C).
ESIMS (negative) m/z 361.5 [MKH]K. 1H NMR
(500 MHz, C5D5N): 7.29 (1H, d, JZ2.1 Hz, H-15), 6.76
(1H, d, JZ2.1 Hz, H-16), 5.43 (1H, br d, JZ4.7 Hz, H-6),
4.15 (1H, ddd, JZ11.9, 9.0, 4.4 Hz, H-2), 3.83 (1H, m,
H-3), 3.27 (1H, ddd, JZ16.8, 10.5, 4.8 Hz, H-12a), 2.97
(1H, ddd, JZ16.8, 10.1, 6.2 Hz, H-12b), 2.83 (1H, td, JZ
11.3, 5.1 Hz, H-8), 2.68 (1H, m, H-7), 2.67 (2H, m, H-4),
2.63 (1H, dd, JZ12.8, 4.3 Hz, H-1b), 2.51 (3H, s, H-21),
2.34 (1H, m, H-7), 2.30 (1H, m, H-11), 2.05 (1H, m, H-11),
3.4.5. Cynanoside E (5). Amorphous powder, [a]D K22.48
(c 1.07, CHCl3, 24 8C). IR (KBr) nmax: 3424, 2925, 1738,
1688, 1068 cmK1. FABMS (positive) m/z: 847 [MCNa]C.
HRFABMS (positive): observed 847.4111, calcd for
1
C42H64O16Na [MCNa]C, 847.4092. H NMR (500 MHz,
C5D5N) and 13C NMR (125 MHz, C5D5N): see Tables 1
and 3.

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5807
1.85 (1H, ddd, JZ11.3, 7.2, 2.0 Hz, H-9), 1.56 (1H, t, JZ
3.7. Enzymatic hydrolysis of 2, 6, and 9
12.3 Hz, H-1a), 1.09 (3H, s, H-19). 13C NMR (125 MHz,
C5D5N): 196.1 (C-13), 179.1 (C-14), 158.0 (C-20), 141.1
(C-15), 140.8 (C-5), 121.6 (C-17), 120.2 (C-6), 110.7
(C-16), 76.7 (C-3), 72.4 (C-2), 48.4 (C-9), 45.8 (C-1), 43.1
(C-8), 42.5 (C-12), 40.6 (C-4), 40.2 (C-10), 30.6 (C-7), 23.7
(C-11), 19.6 (C-19), 14.2 (C-21).
A solution of 2, 6 and 9 (each 1 mg) in 0.1 M acetate buffer
(pH 4.0, 1.0 mL) was treated with naringinase (3 mg, Sigma
Chemical Co.), and then the reaction mixture was stirred at
40 8C for 24 h. The reaction mixture was passed through a
Sep-Pak C18 cartridge using H2O and MeOH. The MeOH
eluate was analyzed by HPLC and TLC to identify the
deglucosyl derivative by comparison with the authentic
samples. HPLC condition: column, YMC-Pack ODS-A,
150!4.6 mm i.d.; flow rate, 0.8 mL/min; column tempera-
ture, 40 8C; tR, 1 5.17 min (with 60% MeOH as eluate
solvent), 5 7.25 min (75% MeOH), 11 7.21 min (80%
MeOH). TLC condition: CHCl3/MeOH (95:5), Rf, 1 0.31; 5
0.40; 11 0.42.
3.5.2. Compound 14. [a]D C14.68 (c 0.26, MeOH, 24 8C).
ESIMS (negative) m/z 391.2 [MKH]K. 1H NMR
(500 MHz, C5D5N): 6.67 (1H, s, H-18), 6.02 (1H, dd, JZ
8.8, 7.4 Hz, H-16), 5.42 (1H, br d, JZ4.4 Hz, H-6), 4.34
(1H, dd, JZ9.3, 7.4 Hz, H-15), 4.15 (1H, ddd, JZ11.7, 9.0,
4.4 Hz, H-2), 4.03 (1H, t, JZ9.1 Hz, H-15), 3.80 (1H, m,
H-3), 2.70 (1H, m, H-7), 2.64 (2H, m, H-4), 2.57 (1H, td,
JZ10.7, 4.4 Hz, H-8), 2.52 (1H, dd, JZ12.9, 4.6 Hz, H-1b),
2.48 (1H, m, H-12), 2.29 (1H, m, H-11), 2.16 (1H, m, H-7),
1.86 (1H, m, H-12), 1.78 (3H, s, H-21), 1.60 (1H, m, H-11),
1.43 (1H, t, JZ10.7 Hz, H-9), 1.39 (1H, t, JZ12.2 Hz,
H-1a), 1.00 (3H, s, H-19). 13C NMR (125 MHz, C5D5N):
175.6 (C-14), 144.6 (C-18), 140.7 (C-5), 120.1 (C-6), 119.8
(C-20), 119.1 (C-13), 92.5 (C-17), 82.0 (C-16), 76.7 (C-3),
72.4 (C-2), 67.2 (C-15), 52.9 (C-9), 45.7 (C-1), 40.6 (C-4),
40.4 (C-8), 40.2 (C-10), 30.7 (C-11), 28.5 (C-7), 20.9
(C-12), 20.7 (C-21), 19.3 (C-19).
The H2O eluate was concentrated to give a residue of the
sugar fraction.
3.8. Methylation of 12, 1 and 3
An excess ethereal solution of diazomethane was added to
12 (1.0 mg) and the mixture was allowed to stand at rt for
1 h. After removal of the solvent by evaporation, the residue
was purified by HPLC with 65% MeOH to give 16 (0.9 mg).
Through a similar procedure, 17 (4.0 mg) and 18 (3.0 mg)
were obtained from 1 (5.2 mg) and 3 (4.1 mg) by HPLC
with 80% and 83% MeOH following methylation with
diazomethane.
3.5.3. Compound 15. [a]D C77.28 (c 0.20, MeOH, 24 8C).
ESIMS (positive) m/z 399.2 [MCNa]C. 1H NMR
(500 MHz, C5D5N): 6.47 (1H, s, H-18), 5.46 (1H, dt, JZ
9.5, 7.3 Hz, H-16), 5.41 (1H, br d, JZ5.0 Hz, H-6), 4.26
(1H, dd, JZ8.4, 7.3 Hz, H-15), 4.16 (1H, ddd, JZ11.8, 8.8,
4.4 Hz, H-2), 3.97 (1H, dd, JZ9.7, 8.6 Hz, H-15), 3.83 (1H,
m, H-3), 3.56 (1H, dd, JZ8.0, 1.3 Hz, H-17), 2.67 (1H, m,
H-7), 2.56–2.65 (4H, m, H-4, H-8 and H-12), 2.51 (1H, dd,
JZ12.5, 4.6 Hz, H-1b), 2.18 (1H, m, H-7), 2.12 (1H, m,
H-11), 1.56 (3H, s, H-21), 1.45 (1H, m, H-11), 1.42 (1H, m,
H-12), 1.41 (1H, t, JZ12.8 Hz, H-1a), 1.38 (1H, t, JZ
10.3 Hz, H-9), 0.98 (3H, s, H-19). 13C NMR (125 MHz,
C5D5N): 175.5 (C-14), 143.8 (C-18), 140.9 (C-5), 120.1
(C-6), 118.6 (C-20), 114.4 (C-13), 76.7 (C-3), 75.6 (C-16),
72.4 (C-2), 67.8 (C-15), 56.2 (C-17), 53.2 (C-9), 45.6 (C-1),
40.6 (C-8), 40.3 (C-10), 40.1 (C-4), 30.1 (C-11), 28.5 (C-7),
24.8 (C-21), 23.9 (C-12), 19.2 (C-19).
3.8.1. Compound 16. ESIMS (positive) m/z 399.2 [MC
Na]C. 1H NMR (500 MHz, C5D5N): 7.43 (1H, d, JZ
2.0 Hz, H-15), 6.81 (1H, d, JZ2.0 Hz, H-16), 5.36 (1H, br
d, JZ4.8 Hz, H-6), 4.13 (1H, ddd, JZ11.7, 8.8, 4.4 Hz, H-
2), 3.82 (1H, m, H-3), 3.70 (3H, s, C-14-OCH3), 2.91 (1H,
ddd, JZ17.0, 10.1, 4.9 Hz, H-12a), 2.83 (1H, ddd, JZ17.0,
9.2, 6.6 Hz, H-12b), 2.67 (1H, td, JZ11.2, 5.4 Hz, H-8),
2.64 (2H, m, H-4), 2.58 (3H, s, H-21), 2.57 (1H, dd, JZ
13.0, 4.4 Hz, H-1b), 2.47 (1H, m, H-7), 2.20 (1H, m, H-11),
2.17 (1H, m, H-7), 1.76 (1H, m, H-11), 1.70 (1H, m, H-9),
1.51 (1H, t, JZ12.3 Hz, H-1a), 1.03 (3H, s, H-19). 13C
NMR (125 MHz, C5D5N): 195.7 (C-13), 176.9 (C-14),
158.1 (C-20), 141.1 (C-5), 141.0 (C-15), 121.5 (C-17),
119.7 (C-6), 110.6 (C-16), 76.6 (C-3), 72.2 (C-2), 51.6
(C-14-OCH3), 48.3 (C-9), 45.6 (C-1), 42.5 (C-8), 42.0
(C-12), 40.5 (C-4), 40.1 (C-10), 30.2 (C-7), 23.2 (C-11),
19.5 (C-19), 14.3 (C-21).
3.6. Acid hydrolysis of 3, 7, 8 and 10
3.8.2. Compound 17. [a]D K31.98 (c 0.40, CHCl3, 24 8C).
ESIMS (positive) m/z 831.5 [MCNa]C. 1H NMR
(500 MHz, C5D5N): 7.42 (1H, d, JZ2.1 Hz, H-15), 6.80
(1H, d, JZ2.1 Hz, H-16), 5.36 (1H, br d, JZ5.0 Hz, H-6),
5.19 (1H, dd, JZ9.6, 1.6 Hz, cym I-H-1), 5.16 (1H, d, JZ
3.4 Hz, dgn-H-1), 5.12 (1H, dd, JZ9.8, 1.7 Hz, cym II-H-
1), 4.22–4.28 (2H, m, cym I-H-5 and dgn-H-5), 4.12 (1H,
dq, JZ9.6, 6.2 Hz, cym II-H-5), 4.09 (1H, br d, JZ1.6 Hz,
dgn-H-4), 4.01 (1H, ddd, JZ11.5, 8.8, 4.3 Hz, H-2), 3.93
(1H, q like, JZ2.8 Hz, cym I-H-3), 3.83 (1H, ddd, JZ12.1,
4.4, 2.7 Hz, dgn-H-3), 3.74 (1H, q like, JZ2.8 Hz, cym II-
H-3), 3.69 (3H, s, C-14-OCH3), 3.62 (1H, ddd, JZ11.2, 8.8,
5.5 Hz, H-3), 3.55–3.56 (4H, cym II-H-4 and cym I-OCH3),
3.47 (3H, s, cym II-OCH3), 3.45–3.46 (4H, cym I-H-4 and
dgn-OCH3), 2.89 (1H, ddd, JZ16.8, 10.1, 5.2 Hz, H-12a),
A solution of 3, 7, 8 and 10 (each 1 mg) in 0.05 M HCl
(dioxane/H2O, 1:1, 200 mL) was heated at 60 8C for 1.5 h.
After dioxane was removed, the solution was extracted with
EtOAc (1 mL!3). Then, the EtOAc extract was analyzed
by HPLC and TLC to identify the aglycon by comparison
with the authentic samples of 12, 14 and 15. HPLC
condition: column, YMC-Pack ODS-A, 150!4.6 mm i.d.;
flow rate, 0.8 mL/min; column temperature, 40 8C; tR, 12
6.46 min (with 50% MeOH as eluate solvent), 14 8.04 min
(50% MeOH), 15 6.59 min (60% MeOH); TLC condition:
CHCl3/MeOH (9:1), Rf, 12 0.21; 14 0.38; 15 0.48.
Subsequently, the aqueous layer was treated with the same
procedure as for 1, 5 and 11.

5808
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
2.82 (1H, ddd, JZ16.8, 9.7, 6.6 Hz, H-12b), 2.64 (1H, td,
JZ11.4, 5.1 Hz, H-8), 2.58 (3H, s, H-21), 2.55 (1H, dd, JZ
13.3, 4.8 Hz, H-1b), 2.41–2.52 (5H, H-4, H-7, cym I-H-2
and cym II-H-2), 2.35 (1H, td, J Z12.6, 4.7 Hz, dgn-H-2a),
2.17 (1H, m, H-11), 2.16 (1H, m, H-7), 2.01 (1H, dd, JZ
12.6, 4.6 Hz, dgn-H-2e), 1.86 (1H, ddd, JZ13.5, 9.8,
2.5 Hz, cym II-H-2a), 1.82 (1H, ddd, JZ13.9, 9.6, 2.0 Hz,
cym I-H-2a), 1.74 (1H, m, H-11), 1.69 (1H, m, H-9), 1.54
(3H, d, JZ6.2 Hz, cym II-H-6), 1.47 (3H, d, JZ6.6 Hz,
dgn-H-6), 1.37 (1H, t, JZ12.4 Hz, H-1a), 1.31 (3H, d, JZ
6.2 Hz, cym I-H-6), 0.98 (3H, s, H-19). 13C NMR
(125 MHz, C5D5N): 195.7 (C-13), 176.8 (C-14), 158.2
(C-20), 141.0 (C-15), 140.1 (C-5), 121.6 (C-17), 120.5
(C-6), 110.7 (C-16), 101.1 (dgn-C-1), 99.5 (cym II-C-1),
97.4 (cym I-C-1), 85.1 (C-3), 82.1 (cym I-C-4), 79.0
(cym II-C-3), 77.5 (cym I-C-3), 74.6 (dgn-C-3), 74.2 (cym
II-C-4), 73.9 (dgn-C-4), 71.1 (cym II-C-5), 69.9 (C-2), 69.6
(cym I-C-5), 67.6 (dgn-C-5), 58.0 (cym II-OCH3), 57.4
(cym I-OCH3), 55.4 (dgn-OCH3), 51.6 (C-14-OCH3), 48.2
(C-9), 44.9 (C-1), 42.4 (C-8), 41.8 (C-12), 39.5 (C-10), 37.6
(C-4), 35.4 (cym II-C-2), 35.2 (cym I-C-2), 32.5 (dgn-C-2),
30.2 (C-7), 23.2 (C-11), 19.2 (C-19), 18.9 (cym II-C-6), 18.4
(cym I-C-6), 17.9 (dgn-C-6), 14.3 (C-21).
3.9. p-Bromobenzoylation of 16, 14 and 15
To a solution of 16 (0.9 mg), 14 (1.2 mg) and 15 (0.8 mg) in
pyridine (100 mL) were added p-bromobrnzoyl chloride
(5 mg) and N,N-dimethylamine pyridine (1 mg). The
mixture was allowed to stand at rt for 24 h. After
evaporation of the solvent, the residue was further purified
by HPLC with 97% MeOH to give 19 (0.9 mg), 20 (1.1 mg)
and 21 (0.4 mg).
3.9.1. Compound 19. UV (MeOH) lmax nm (log 3): 245
(4.56). CD (MeOH) D3: K31.69 at 254 nm, C19.13 at
1
237 nm. H NMR (500 MHz, CDCl3): 7.20 (1H, d, JZ
2.1 Hz, H-15), 6.58 (1H, d, JZ2.1 Hz, H-16), 5.51–5.58
(2H, m, H-2 and H-6), 5.14 (1H, ddd, JZ11.8, 9.7, 5.5 Hz,
H-3), 3.69 (3H, s, C-14-OCH3), 2.75 (1H, ddd, JZ17.2,
10.8, 4.8 Hz, H-12a), 2.54–2.64 (4H, m, H-4, H-8 and
H-12b), 2.51 (3H, s, H-21), 2.40 (1H, dd, JZ12.7, 4.8 Hz,
H-1b), 2.46 (1H, m, H-7), 2.20 (1H, m, H-7), 1.97 (1H, m,
H-11), 1.58–1.63 (2H, m, H-9 and H-11), 1.40 (1H, t, JZ
12.4 Hz, H-1a), 1.23 (3H, s, H-19).
3.9.2. Compound 20. UV (MeOH) lmax nm (log 3): 245
(4.67). CD (MeOH) D3: K39.04 at 254 nm, C32.90 at
238 nm. H NMR (500 MHz, CDCl3): 6.37 (1H, s, H-18),
1
3.8.3. Compound 18. [a]D K55.68 (c 0.30, CHCl3, 24 8C).
ESIMS (positive) m/z 817.5 [MCNa]C. HRFABMS
(positive): observed 817.4006, calcd for C41H62O15Na
5.59 (1H, br d, JZ5.3 Hz, H-6), 5.51 (1H, ddd, JZ11.9, 9.9,
4.6 Hz, H-2), 5.38 (1H, dd, JZ10.0, 7.2 Hz, H-16), 5.17
(1H, ddd, JZ11.6, 9.9, 5.6 Hz, H-3), 4.19 (1H, dd, JZ10.0,
7.2 Hz, H-15), 3.85 (1H, dd, JZ9.9, 9.1 Hz, H-15), 2.70
(1H, dd, JZ13.7, 5.5 Hz, H-4a), 2.50–2.57 (4H, m, H-4b,
H-7, H-8 and H-12), 2.46 (1H, dd, JZ12.6, 4.8 Hz, H-1b),
2.11–2.18 (2H, m, H-7 and H-11), 1.48 (3H, s, H-21), 1.36–
1.46 (3H, m, H-9, H-11 and H-12), 1.39 (1H, t, JZ12.3 Hz,
H-1a), 1.15 (3H, s, H-19).
1
[MCNa]C, 817.3987. H NMR (500 MHz, C5D5N): 7.41
(1H, d, JZ2.1 Hz, H-15), 6.80 (1H, d, JZ2.1 Hz, H-16),
5.37 (1H, br d, JZ5.0 Hz, H-6), 5.23 (1H, dd, JZ9.6,
1.8 Hz, dgt-H-1), 5.18 (1H, dd, JZ9.7, 1.6 Hz, b-cym-H-1),
5.09 (1H, dd, JZ3.9 2.5 Hz, a-cym-H-1), 4.45–4.50 (2H, m,
a-cym-H-5 and dgt-H-3), 4.21 (1H, dq, JZ9.7, 6.2 Hz,
b-cym-H-5), 4.13 (1H, dq, JZ9.6, 6.2 Hz, dgt-H-5), 4.07
(1H, q like, JZ2.8 Hz, b-cym-H-3), 3.99 (1H, ddd, JZ11.7,
8.7, 4.6 Hz, H-2), 3.72 (1H, q like, JZ2.6 Hz, a-cym-H-3),
3.69 (3H, s, C-14-OCH3), 3.62 (1H, m, a-cym-H-4), 3.62
(3H, s, b-cym-OCH3), 3.59 (1H, ddd, JZ11.2, 8.7, 5.5 Hz,
H-3), 3.47 (1H, dd, JZ9.7, 2.8 Hz, b-cym-H-4), 3.43 (1H,
dd, JZ9.6, 3.1 Hz, dgt-H-4), 3.40 (3H, s, a-cym-OCH3),
2.89 (1H, ddd, JZ16.8, 9.8, 4.8 Hz, H-12a), 2.82 (1H, ddd,
JZ16.8, 9.2, 6.8 Hz, H-12b), 2.64 (1H, td, JZ11.2, 4.8 Hz,
H-8), 2.58 (3H, s, H-21), 2.54 (1H, dd, JZ13.1, 4.6 Hz,
H-1b), 2.45–2.51 (3H, m, H-4 and H-7), 2.36–2.41 (2H, m,
a-cym-H-2e and dgt-H-2e), 2.34 (1H, ddd, JZ13.5, 3.4,
2.1 Hz, b-cym-H-2e), 2.17 (1H, m, H-11), 2.16 (1H, m,
H-7), 1.91 (1H, ddd, JZ13.5, 9.6, 2.7 Hz, dgt-H-2a), 1.84–
1.88 (2H, m, b-cym-H-2a and a-cym-H-2a), 1.74 (1H, m,
H-11), 1.69 (1H, m, H-9), 1.44 (3H, d, JZ6.4 Hz, a-cym-H-
6), 1.39 (3H, d, JZ6.2 Hz, dgt-H-6), 1.35 (1H, t, JZ
12.4 Hz, H-1a), 1.29 (3H, d, JZ6.2 Hz, b-cym-H-6), 0.96
(3H, s, H-19). 13C NMR (125 MHz, C5D5N): 195.7 (C-13),
176.8 (C-14), 158.2 (C-20), 141.0 (C-15), 140.0 (C-5),
121.5 (C-17), 120.4 (C-6), 110.7 (C-16), 100.5 (dgt-C-1),
98.5 (a-cym-C-1), 97.8 (b-cym-C-1), 85.2 (C-3), 82.9
(b-cym-C-4), 80.8 (dgt-C-4), 77.9 (b-cym-C-3), 76.6
(a-cym-C-3), 72.7 (a-cym-C-4), 69.9 (C-2), 69.4 (b-cym-
C-5), 68.9 (dgt-C-5), 67.8 (dgt-C-3), 67.3 (a-cym-C-5), 59.0
(b-cym-OCH3), 56.8 (a-cym-OCH3), 51.6 (C-14-OCH3),
48.2 (C-9), 44.8 (C-1), 42.4 (C-8), 41.9 (C-12), 39.5 (C-10),
38.5 (dgt-C-2), 37.6 (C-4), 37.0 (b-cym-C-2), 32.3 (a-cym-
C-2), 30.2 (C-7), 23.2 (C-11), 19.2 (C-19), 18.5 (dgn-C-6),
18.4 (a-cym-C-6), 18.2 (b-cym-C-6), 14.3 (C-21).
3.9.3. Compound 21. UV (MeOH) lmax nm (log 3): 244
(4.67). CD (MeOH) D3: K35.24 at 253 nm, C29.76 at
238 nm. H NMR (500 MHz, CDCl3): 6.24 (1H, s, H-18),
1
5.59 (1H, br d, JZ5.5 Hz, H-6), 5.51 (1H, ddd, JZ11.9, 9.7,
4.7 Hz, H-2), 5.32 (1H, dt, JZ9.6, 7.1 Hz, H-16), 5.16 (1H,
ddd, JZ11.7, 9.7, 5.9 Hz, H-3), 4.17 (1H, dd, JZ8.6,
7.1 Hz, H-15), 3.86 (1H, t, JZ9.3 Hz, H-15), 3.44 (1H, dd,
JZ8.6, 1.1 Hz, H-17), 2.69 (1H, dd, JZ13.7, 5.9 Hz,
H-4a), 2.48–2.58 (4H, m, H-4b, H-7, H-8 and H-12), 2.43
(1H, dd, JZ12.8, 4.6 Hz, H-1b), 2.06–2.14 (2H, m, H-7 and
H-11), 1.30–1.54 (4H, m, H-1a, H-9, H-11 and H-12), 1.26
(3H, s, H-21), 1.15 (3H, s, H-19).
3.10. Preparation of the (R)-MPTA ester and (S)-MPTA
ester from 17, 18, 5 and 8
(S)- and (R)-a-methoxy-a-(trifluoromethyl)-phenylacetyl
chloride (10 mL each) were added to the solution of 17
(0.9 mg) in pyridine (100 mL). After being stirred at rt for
4 h, the mixture was evaporated to dryness and purified by
HPLC with 92% MeOH to give (R)-MPTA ester 22
(1.0 mg) and (S)-MPTA ester 23 (0.9 mg). Using a similar
procedure, (R)-MPTA ester [24 (1.0 mg), 26 (4.9 mg) and
28 (1.2 mg)] and (S)-MPTA ester [25 (1.0 mg), 27 (4.9 mg)
and 29 (1.2 mg)] were obtained from 18 (1.1 mg), 5
(5.0 mg) and 8 (1.3 mg), respectively.
1
3.10.1. Compound 22. H NMR (500 MHz, CDCl3): 7.22

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5809
(1H, d, JZ2.0 Hz, H-15), 6.55 (1H, d, JZ2.0 Hz, H-16),
5.44 (1H, br d, JZ5.3 Hz, H-6), 5.30 (1H, ddd, JZ12.0, 9.6,
4.7 Hz, H-2), 4.98 (1H, d, JZ3.7 Hz, dgn-H-1), 4.86 (1H,
dd, JZ9.4, 1.8 Hz, cym I-H-1), 4.82 (1H, dd, JZ9.6,
1.9 Hz, cym II-H-1), 4.72 (1H, dd, JZ9.8, 3.2 Hz, cym
II-H-4), 3.95–3.99 (3H, m, dgn-H-5, cym II-H-3 and cym
II-H-5), 3.86 (2H,m, dgn-H-4 and H-3), 3.78 (1H, dq, JZ
9.6, 6.2 Hz, cym I-H-5), 3.69 (1H, m, cym I-H-3), 3.67 (3H,
s, C-14-OCH3), 3.60 (1H, m, dgn-H-3), 3.40 (3H, s, cym
I-OCH3), 3.39 (3H, s, dgn-OCH3), 3.35 (3H, s, cym
II-OCH3), 3.15 (1H, dd, JZ9.6, 2.9 Hz, cym I-H-4), 2.66
(1H, dd, JZ17.0, 10.3, 5.1 Hz, H-12a), 2.55 (3H, s, H-21),
2.53–2.56 (3H, m, H-4a, H-8 and H-12b), 2.39 (1H, ddd,
JZ14.2, 3.4, 1.8 Hz, cym II-H-2e), 2.31 (1H, m, H-7), 2.28
(1H, m, H-4b), 2.18 (1H, dd, JZ12.8, 5.1 Hz, H-1b), 2.17
(1H, m, H-7), 2.16 (1H, ddd, JZ13.8, 3.5, 2.3 Hz, cym I-H-
2e), 2.03 (1H, td, J Z12.8, 3.9 Hz, dgn-H-2a), 1.90 (1H, m,
H-11), 1.87 (1H, m, cym II-H-2a), 1.83 (1H, m, dgn-H-2e),
1.48–1.53 (3H, m, H-9, H-11 and cym I-H-2a), 1.24 (3H, d,
JZ6.6 Hz, dgn-H-6), 1.17 (1H, t, JZ12.4 Hz, H-1a), 1.12
(3H, s, H-19), 1.12 (3H, d, JZ6.2 Hz, cym I-H-6), 1.10 (3H,
d, JZ6.2 Hz, cym II-H-6).
dd, JZ12.6, 4.8 Hz, H-1b), 2.16 (1H, m, H-7), 2.09–2.14
(2H, m, a-cym-H-2e and dgt-H-2e), 2.06 (1H, ddd, JZ13.8,
3.2, 2.2 Hz, b-cym-H-2e), 1.92 (1H, ddd, JZ14.2, 9.6,
2.3 Hz, dgt-H-2a), 1.90 (1H, m, H-11), 1.84 (1H, m, a-cym-
H-2a), 1.47–1.51 (3H, m, H-9, H-11 and b-cym-H-2a), 1.24
(3H, d, JZ6.2 Hz, dgt-H-6), 1.21 (3H, d, JZ6.4 Hz, a-cym-
H-6), 1.16 (1H, t, JZ12.4 Hz, H-1a), 1.12 (3H, s, H-19),
1.04 (3H, d, JZ6.1 Hz, b-cym-H-6).
1
3.10.4. Compound 25. H NMR (500 MHz, CDCl3): 7.21
(1H, d, JZ2.1 Hz, H-15), 6.57 (1H, d, JZ2.1 Hz, H-16),
5.58 (1H, q like, JZ2.8 Hz, dgt-H-3), 5.44 (1H, br d, JZ
5.3 Hz, H-6), 5.30 (1H, ddd, JZ11.9, 9.7, 4.8 Hz, H-2), 4.89
(1H, dd, JZ4.7, 2.7 Hz, a-cym-H-1), 4.80 (1H, dd, JZ8.7,
3.4 Hz, a-cym-H-4), 4.70 (dd, JZ9.6, 1.9 Hz, b-cym-H-1),
4.24–4.28 (2H, m, dgt-H-1 and a-cym-H-5), 3.80–3.86 (2H,
m, dgt-H-5 and H-3), 3.83 (1H, m, a-cym-H-3), 3.77 (1H,
m, dgt-H-5), 3.79 (1H, ddd, JZ11.7, 9.7, 5.0, H-3), 3.68
(3H, s, C-14-OCH3), 3.58–3.61 (2H, m, b-cym-H-3 and
b-cym-H-5), 3.37 (3H, s, b-cym-OCH3), 3.29 (1H, dd, JZ
9.5, 2.8 Hz, dgt-H-4), 3.24 (3H, s, a-cym-OCH3), 2.60–3.68
(3H, m, b-cym-H-4 and H-12), 2.56 (3H, s H-21), 2.54 (1H,
m, H-8), 2.52 (1H, dd, JZ13.7, 5.0 Hz, H-4a), 2.34 (1H, m,
H-7), 2.29 (1H, dd, JZ12.8, 4.8 Hz, H-1b), 2.24 (1H, m,
H-4b), 2.20 (1H, m, a-cym-H-2e), 2.14 (1H, m, H-7), 1.89–
1.97 (4H, m, H-11, b-cym-H-2e, dgt-H-2e and a-cym-H-
2a), 1.80 (1H, ddd, JZ14.2, 9.6, 2.3 Hz, dgt-H-2a), 1.49–
1.60 (2H, m, H-9 and H-11), 1.31 (1H, t, JZ12.6 Hz, H-1a),
1.28 (1H, ddd, JZ13.8, 9.6, 2.5 Hz, b-cym-H-2a), 1.25 (3H,
d, JZ6.2 Hz, dgt-H-6), 1.12 (3H, s, H-19), 1.12 (3H, d, JZ
6.2 Hz, a-cym-H-6), 0.76 (3H, d, JZ6.2 Hz, b-cym-H-6).
1
3.10.2. Compound 23. H NMR (500 MHz, CDCl3): 7.22
(1H, d, JZ2.0 Hz, H-15), 6.57 (1H, d, JZ2.0 Hz, H-16),
5.45 (1H, br d, JZ5.0 Hz, H-6), 5.32 (1H, ddd, JZ11.9, 9.8,
4.8 Hz, H-2), 4.91 (1H, d, JZ3.5 Hz, dgn-H-1), 4.80 (1H,
dd, JZ9.6, 1.7 Hz, cym II-H-1), 4.76 (1H, dd, JZ9.4,
1.6 Hz, cym I-H-1), 4.75 (1H, dd, JZ9.7, 3.1 Hz, cym II-H-
4), 4.01 (1H, dq, JZ9.9, 6.2 Hz, cym II-H-5), 3.93 (1H, q,
JZ6.5 Hz, dgn-H-5), 3.82–3.85 (2H, m, dgn-H-4 and cym
II-H-3), 3.80 (1H, ddd, JZ11.4, 9.8, 5.5 Hz, H-3), 3.68 (3H,
s, C-14-OCH3), 3.67 (1H, m, cym I-H-5), 3.60 (1H, m, cym
I-H-3), 3.58 (1H, ddd, JZ12.4, 4.3, 2.5 Hz, dgn-H-3), 3.39
(3H, s, cym I-OCH3), 3.38 (3H, s, dgn-OCH3), 3.17 (3H, s,
cym II-OCH3), 2.96 (1H, dd, JZ9.6, 3.0 Hz, cym I-H-4),
2.66 (2H, m, H-12), 2.55 (3H, s, H-21), 2.54 (1H, m, H-8),
2.52 (1H, dd, JZ13.7, 5.5 Hz, H-4a), 2.26–2.35 (4H, m,
H-1b, H-4b, H-7 and cym II-H-2e), 2.16 (1H, m, H-7), 2.07
(1H, ddd, JZ13.8, 3.5, 1.8 Hz, cym I-H-2e), 2.02 (1H, td, J
Z12.6, 4.6 Hz, dgn-H-2a), 1.97 (1H, m, H-11), 1.86 (1H, m,
cym II-H-2a), 1.82 (1H, dd, JZ12.6, 4.6 Hz, dgn-H-2e),
1.57 (1H, m, H-11), 1.54 (1H, m, H-9), 1.36 (1H, ddd, JZ
13.8, 9.4, 2.1 Hz, cym I-H-2a), 1.32 (1H, t, JZ12.6 Hz,
H-1a), 1.22 (3H, d, J Z6.6 Hz, dgn-H-6), 1.21 (3H, d, JZ
6.4 Hz, cym II-H-6), 1.13 (3H, s, H-19), 0.98 (3H, d, JZ
6.2 Hz, cym I-H-6).
1
3.10.5. Compound 26. H NMR (500 MHz, CDCl3): 6.39
(1H, s, H-18), 5.48 (1H, br d, JZ5.1 Hz, H-6), 5.36 (1H, dd,
JZ10.0, 7.2 Hz, H-16), 5.27 (1H, ddd, JZ11.9, 9.6, 4.7 Hz,
H-2), 4.96 (1H, d, JZ3.4 Hz, dgn-H-1), 4.85 (1H, dd, JZ
9.4, 1.6 Hz, cym I-H-1), 4.81 (1H, dd, JZ9.6, 1.7 Hz, cym
II-H-1), 4.71 (1H, dd, JZ9.9, 3.2 Hz, cym II-H-4), 4.16
(1H, dd, JZ8.9, 7.2 Hz, H-15a), 3.98–3.99 (3H, m, dgn-H-5,
cym II-H-3 and cym II-H-5), 3.84–3.88 (2H, m, H-3 and
dgn-H-4), 3.83 (1H, t, JZ9.9 Hz, H-15b), 3.77 (1H, dq, JZ
9.6, 6.4 Hz, cym I-H-5), 3.68 (1H, q like, JZ2.9 Hz, cym
I-H-3), 3.59 (1H, m, dgn-H-3), 3.40 (3H, s, cym I-OCH3),
3.39 (3H, s, dgn-OCH3), 3.34 (3H, s, cym II-OCH3), 3.14
(1H, dd, JZ9.6, 2.9 Hz, cym I-H-4), 2.55 (1H, dd, JZ13.7,
5.4 Hz, H-4a), 2.48, (1H, m, H-12), 2.42–2.46 (2H, m, H-7
and H-8), 2.39 (1H, ddd, JZ14.2, 3.8, 1.9 Hz, cym II-H-2e),
2.25 (1H, m, H-4b), 2.16 (1H, dd, JZ12.9, 5.1 Hz, H-1b),
2.14 (1H, ddd, JZ14.2, 3.5, 2.1 Hz, cym I-H-2e), 2.05–2.08
(2H, m, H-7 and H-11), 2.03 (1H, td, JZ12.6, 4.3 Hz, dgn-
H-2a), 1.87 (1H, ddd, JZ14.2, 9.6, 2.5 Hz, cym II-H-2a),
1.82 (1H, dd, JZ12.6, 4.6 Hz, dgn-H-2e), 1.48 (1H, ddd,
JZ14.2, 9.4, 2.4 Hz, cym I-H-2a), 1.47 (3H, s, H-21), 1.38
(1H, m, H-12), 1.33 (1H, m, H-11), 1.30 (1H, m, H-9), 1.24
(3H, d, JZ6.6 Hz, dgn-H-6), 1.15 (1H, t, JZ12.4 Hz,
H-1a), 1.10 (3H, d, JZ6.4 Hz, cym I-H-6), 1.09 (3H, d, JZ
6.4 Hz, cym II-H-6), 1.03 (3H, s, H-19).
1
3.10.3. Compound 24. H NMR (500 MHz, CDCl3): 7.21
(1H, d, JZ2.1 Hz, H-15), 6.55 (1H, d, JZ2.1 Hz, H-16),
5.59 (1H, q like, JZ2.8 Hz, dgt-H-3), 5.45 (1H, br d, JZ
5.3 Hz, H-6), 5.28 (1H, ddd, JZ11.9, 9.7, 4.8 Hz, H-2), 4.86
(br s, a-cym-H-1), 4.85 (dd, JZ9.6, 2.2 Hz, b-cym-H-1),
4.78 (1H, dd, JZ8.7, 3.2 Hz, a-cym-H-4), 4.70 (1H, dd, JZ
9.6, 1.7 Hz, dgt-H-1), 4.26 (1H, dq, JZ8.7, 6.4 Hz, a-cym-
H-5), 3.80–3.86 (2H, m, dgt-H-5 and H-3), 3.75–3.79 (2H,
m, b-cym-H-3 and b-cym-H-5), 3.71 (1H, m, a-cym-H-3),
3.67 (3H, s, C-14-OCH3), 3.43 (3H, s, b-cym-OCH3), 3.29
(1H, dd, JZ9.5, 2.8 Hz, dgt-H-4), 3.07 (1H, d, JZ9.6,
3.0 Hz, b-cym-H-4), 3.07 (3H, s, a-cym-OCH3), 2.66 (1H,
ddd, JZ16.9, 10.1, 5.0 Hz, H-12), 2.57 (1H, dd, JZ14.0,
5.2 Hz, H-4a), 2.55 (3H, s H-21), 2.54 (1H, m, H-12), 2.52
(1H, m, H-8), 2.23–2.35 (2H, m, H-4b and H-7), 2.18 (1H,
1
3.10.6. Compound 27. H NMR (500 MHz, CDCl3): 6.40
(1H, s, H-18), 5.49 (1H, br d, JZ5.3 Hz, H-6), 5.37 (1H, dd,
JZ9.9, 7.1 Hz, H-16), 5.30 (1H, ddd, JZ11.9, 9.6, 4.6 Hz,
H-2), 4.89 (1H, d, JZ3.7 Hz, dgn-H-1), 4.80 (1H, dd, JZ
9.6, 1.7 Hz, cym II-H-1), 4.74 (1H, dd, JZ9.8, 3.0 Hz, cym

5810
H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
II-H-4), 4.74 (1H, dd, JZ9.8, 2.1 Hz, cym I-H-4), 4.17 (1H,
dd, JZ9.2, 7.1 Hz, H-15a), 4.01 (1H, dq, JZ9.6, 6.2 Hz,
cym II-H-5), 3.93 (1H, q, JZ6.6 Hz, dgn-H-5), 3.84 (1H, t,
JZ9.2 Hz, H-15b), 3.83–3.86 (2H, m, dgn-H-4 and cym
II-H-3), 3.80 (1H, ddd, JZ11.6, 9.6, 5.2 Hz, H-3), 3.67 (1H,
dq, JZ9.6, 6.4 Hz, cym I-H-5), 3.60 (1H, m, cym I-H-3),
3.58 (1H, ddd, JZ12.6, 4.8, 2.9 Hz, dgn-H-3), 3.39 (3H, s,
cym I-OCH3), 3.38 (3H, s, dgn-OCH3), 3.17 (3H, s, cym
II-OCH3), 2.92 (1H, dd, JZ9.6, 3.0 Hz, cym I-H-4), 2.55
(1H, m, H-12), 2.52 (1H, dd, JZ13.9, 5.4 Hz, H-4a), 2.45–
2.47 (2H, m, H-7 and H-8), 2.32 (1H, ddd, JZ14.3, 3.9,
2.1 Hz, cym II-H-2e), 2.26 (1H, m, H-1b), 2.25 (1H, m,
H-4b), 2.05–2.15 (3H, m, H-7, H-11 and cym I-H-2e), 2.01
(1H, td, JZ12.6, 3.9 Hz, dgn-H-2a), 1.86 (1H, ddd, JZ
14.3, 9.6, 2.5 Hz, cym II-H-2a), 1.82 (1H, dd, JZ12.6,
4.5 Hz, dgn-H-2e), 1.48 (3H, s, H-21), 1.42 (1H, m, H-12),
1.32–1.37 (3H, m, H-9, H-11 and cym I-H-2a), 1.30 (1H, t,
JZ12.4 Hz, H-1a), 1.22 (3H, d, JZ6.6 Hz, dgn-H-6), 1.21
(3H, d, JZ6.2 Hz, cym II-H-6), 1.05 (3H, s, H-19), 0.94
(3H, d, JZ6.4 Hz, cym I-H-6).
1.71 (1H, ddd, JZ13.1, 11.6, 4.0 Hz, ole-H-2a), 1.48 (3H, s,
H-21), 1.43 (1H, m, H-12), 1.26–1.33 (4H, m, H-1a, H-9,
H-11 and cym-H-2a), 1.29 (3H, d, JZ6.2 Hz, dgt-H-6), 1.13
(3H, d, JZ6.2 Hz, ole-H-6), 1.04 (3H, s, H-19), 0.75 (3H, d,
JZ6.4 Hz, cym-H-6).
3.11. Chemical conversion of cynanoside D (4) into
cynanoside B (2)
Treatment of 4 (0.5 mg) with 0.3% NaOH (25 mL) and
acetone (25 mL) at rt overnight afforded 2, which was
detected on the basis of HPLC analysis [condition: column,
CAPCELL PAK C18, 250 ! 4.6 mm i.d.; flow rate, 0.8 mL/
min; column temperature, 40 8C; tR, 7.16 min (with 35%
CH3CN as eluate solvent)] and TLC examination [CHCl3/
MeOH/H2O (60:15:1), Rf, 0.30].
3.12. Absolute configuration of sugars in 1–10
The sugar fractions obtained by acid or enzymatic
hydrolysis as described above were analyzed by HPLC
under the following conditions: column, Shodex SC-1101,
300!8 mm i.d; flow rate, 1 mL/min; column temperature,
80 8C; solvent, H2O; detection, RI (Shodex RI-101) and
OR (Shodex OR-2) detector. Identification of D-cymarose,
L-dignose, D-digitoxose and D-glucose in each sugar fraction
was carried out by a comparison of the retention times and
polarities with those of authentic samples. tR, (min) 8.00
(D-glucose, positive polarity), 9.19 (D-cymarose, positive
polarity), 9.46 (L-diginose, negative polarity) and 9.55
(D-digitoxose, positive polarity). D-cymarose and L-diginose
were detected from 1 and 5. Cymarose (a mixture of D- and
L-form in the ratio 1:1) and D-digitoxose were detected from
3, 7 and 10. D-Cymarose and D-digitoxose were detected
from 8. D-Glucose was detected from 2, 6 and 9.
1
3.10.7. Compound 28. H NMR (500 MHz, CDCl3): 6.40
(1H, s, H-18), 5.52 (1H, q like, 2.8 Hz, dgt-H-3), 5.49 (1H,
br d, JZ5.2 Hz, H-6), 5.37 (1H, dd, JZ9.8, 7.1 Hz, H-16),
5.25 (1H, ddd, JZ11.7, 9.4, 4.6 Hz, H-2), 4.98 (br d, JZ
3.2 Hz, ole-H-1), 4.83 (t, JZ9.6 Hz, ole-H-4), 4.83 (1H, m,
cym-H-1), 4.54 (1H, dd, JZ9.7, 1.9 Hz, dgt-H-1), 4.17 (1H,
dd, JZ9.0, 7.1 Hz, H-15a), 3.97 (1H, dq, JZ9.6, 6.2 Hz,
ole-H-5), 3.85 (1H, m, H-3), 3.83 (1H, t, JZ9.6 Hz, H-15b),
3.76 (1H, dq, JZ9.6, 6.1 Hz, cym-H-5), 3.74 (1H, m, cym-
H-3), 3.73 (1H, dq, JZ9.7, 6.2 Hz, dgt-H-5), 3.43 (1H, m,
ole-3), 3.42 (3H, s, cym-OCH3), 3.39 (1H, dd, JZ9.7,
2.8 Hz, dgt-H-4), 3.09 (3H, s, ole-OCH3), 3.02 (1H, dd, JZ
9.6, 2.8 Hz, cym-H-4), 2.57 (1H, dd, JZ14.0, 5.3 Hz,
H-4a), 2.50 (1H, m, H-12), 2.40–2.46 (2H, m, H-7 and H-8),
2.27 (1H, dd, JZ12.4, 5.1 Hz, ole-H-2e), 2.24 (1H, m,
H-4b), 2.18 (1H, dd, JZ12.7, 5.0 Hz, H-1b), 2.03–2.09 (4H,
m, H-7, H-11, cym-H-2e and dgt-H-2e), 1.92 (1H, ddd, JZ
14.4, 9.7, 2.5 Hz, dgt-H-2a), 1.64 (1H, ddd, JZ13.1, 11.7,
3.9 Hz, ole-H-2a), 1.48 (3H, s, H-21), 1.43 (1H, m, cym-H-
2a), 1.38 (1H, m, H-12), 1.24–1.30 (2H, m, H-9 and H-11),
1.24 (3H, d, JZ6.2 Hz, dgt-H-6), 1.24 (3H, d, JZ6.2 Hz,
ole-H-6), 1.14 (1H, t, JZ12.4 Hz, H-1a), 1.03 (3H, s,
H-19), 0.99 (3H, d, JZ6.1 Hz, cym-H-6).
Acknowledgements
We are grateful to Prof. Susumu Kitanaka and Dr. Liyan
Wang, College of Pharmacy, Nihon University, for their
technical assistance during determination of the absolute
configuration of the sugars.
1
3.10.8. Compound 29. H NMR (500 MHz, CDCl3): 6.40
(1H, s, H-18), 5.52 (1H, q like, 2.8 Hz, dgt-H-3), 5.49 (1H,
br d, JZ5.4 Hz, H-6), 5.37 (1H, dd, JZ9.9, 7.1 Hz, H-16),
5.29 (1H, ddd, JZ11.9, 9.6, 4.5 Hz, H-2), 5.05 (br d, JZ
3.4 Hz, ole-H-1), 4.86 (t, JZ9.7 Hz, ole-H-4), 4.71 (1H, dd,
JZ9.6, 1.8 Hz, cym-H-1), 4.41 (1H, dd, JZ9.6, 1.8 Hz, dgt-
H-1), 4.17 (1H, dd, JZ9.9, 7.1 Hz, H-15a), 3.90 (1H, dq,
JZ9.7, 6.2 Hz, ole-H-5), 3.84 (1H, dd, JZ9.8, 9.1 Hz,
H-15b), 3.80 (1H, dq, JZ9.6, 6.2 Hz, dgt-H-5), 3.77 (1H,
ddd, JZ11.5, 9.6, 5.5 Hz, H-3), 3.59–3.65 (2H, m, cym-H-3
and cym-H-5), 3.48 (1H, m, ole-H-3), 3.43 (1H, dd, JZ9.6,
2.8 Hz, dgt-H-4), 3.38 (3H, s, cym-OCH3), 3.26 (3H, s, ole-
OCH3), 2.73 (1H, dd, JZ9.9, 2.9 Hz, cym-H-4), 2.54 (1H,
m, H-12), 2.52 (1H, dd, JZ13.8, 5.3 Hz, H-4a), 2.45–2.46
(2H, m, H-7 and H-8), 2.35 (1H, dd, JZ12.6, 4.7 Hz, ole-H-
2e), 2.26 (1H, dd, JZ12.9, 4.9 Hz, H-1b), 2.22 (1H, m,
H-4b), 2.11 (1H, m, H-11), 2.10 (1H, m, H-7), 1.94–1.97
(2H, m, cym-H-2e and dgt-H-2e), 1.85 (1H, m, dgt-H-2a),
References and notes
1. (a) Zhang, R. S.; Ye, Y. P.; Shen, Y. M.; Liang, H. L.
Tetrahedron 2000, 56, 3875–3879. (b) Mellado, G. G.; Zubia,
E.; Ortega, M. J.; Lopez-Gonzalez, P. J. Steroids 2004, 69,
291–299. (c) Halaweish, F. T.; Huntimer, E.; Khalil, A. T.
Phytochem. Anal. 2004, 15, 189–194.
2. Yin, J.; Kouda, K.; Tezuka, Y.; Tran, Q. L.; Miyahara, T.;
Chen, Y. J.; Kadota, S. J. Nat. Prod. 2003, 66, 646–650.
3. Liu, H. W.; Xiong, Z.; Li, F.; Qu, G. X.; Kobayashi, H.; Yao,
X. S. Chem. Pharm. Bull. 2003, 51, 1089–1091.
4. Deepak, D.; Srivastav, S.; Khare, A. In Herz, W., Kirby, G. W.,
Moore, R. E., Strglich, W., Tamm, Ch., Eds.; Progress in the
chemistry of organic natural products; Springer: New York,
1997; Vol. 71, pp 169–325.

H. Bai et al. / Tetrahedron 61 (2005) 5797–5811
5811
5. Shanghai Scientific Technological Publishers, Eds.; Zhong
Hua Ben Cao; 1985, Vol. 6, pp 330–333.
S.; Hayashi, K.; Kaneko, K.; Mitsuhashi, H. i.; Snyckers, F. O.;
Fourie, T. G. Chem. Pharm. Bull. 1986, 34, 1337–1339. (d)
Abe, F.; Hirokawa, M.; Yamauchi, T.; Honda, K.; Hayashi, N.;
Nishida, R. Chem. Pharm. Bull. 1999, 47, 1384–1387.
10. (a) Nakagawa, T.; Hayashi, K.; Mitsuhashi, H. Chem. Pharm.
Bull. 1983, 31, 2244–2253. (b) Nakagawa, T.; Hayashi, K.;
Wada, K.; Mitsuhashi, H. Tetrahedron 1983, 39, 607–612. (c)
Qiu, S. X.; Zhang, Z. X.; Yong, L.; Zhou, J. Planta Med. 1991,
57, 454–456. (d) Wang, L. Q.; Shen, Y. M.; Xu, X.; Wei,
Y. Q.; Zhou, J. Steroids 2004, 69, 319–324.
6. (a) Zhang, Z. X.; Zhou, J.; Hayashi, K.; Mitsuhashi, H. Chem.
Pharm. Bull. 1985, 33, 1507–1514. (b) Zhang, Z. X.; Zhou, J.;
Hayashi, K.; Mitsuhashi, H. Chem. Pharm. Bull. 1985, 33,
4188–4192. (c) Zhang, Z. X.; Zhou, J.; Hayashi, K.; Kaneko,
K. Phytochemistry 1988, 27, 2935–2941. (d) Day, S. H.;
Wang, J. P.; Won, S. J.; Lin, C. N. J. Nat. Prod. 2001, 64,
608–611. (e) Lee, K. Y.; Sung, H.; Kim, Y. C. Helv. Chim.
Acta 2003, 86, 474–483.
7. Harada, N.; Ohashi, M.; Nakanishi, K. J. Am. Chem. Soc.
1968, 90, 7349–7351.
11. Konda, Y.; Toda, Y.; Takayanagi, H.; Ogura, H.; Harigaya, Y.;
Lou, H. X.; Li, X.; Onda, M. J. Nat. Prod. 1992, 55,
1118–1123.
8. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092–4096.
12. Sargent, J. M.; Taylor, C. G. Br. J. Cancer 1989, 60, 206–211.
13. Yeo, H.; Kim, K. W.; Kim, J.; Choi, Y. H. Phytochemistry
1998, 49, 1129–1133.
9. (a) Deepak, D.; Khare, M. P.; Khare, A. Phytochemistry 1985,
24, 3015–3017. (b) Tsukamoto, S.; Hayashi, K.; Mitsuhashi,
H. Chem. Pharm. Bull. 1985, 33, 2294–2304. (c) Tsukamoto,