Twelve pregnane glycosides from Cynanchum atratum

Article abstract of DOI:10.1016/j.steroids.2008.10.007

Eleven new 14,15-seco-pregnane-type steroidal glycosides, cynanosides P₁-P₅, Q₁-Q₃, R₁-R₃, and a novel 12,13-seco-14,18-nor-pregnane-type steroidal glycoside, cynanoside S, were isolated fr

Full text of DOI:10.1016/j.steroids.2008.10.007

Steroids 74 (2009) 198–207
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Twelve pregnane glycosides from Cynanchum atratum
Hong Baia,b, Wei Li , Yoshihisa Asada , Tadaaki Satou , Yuanshu Wang , Kazuo Koike
a,∗
c
a
b
a
a
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
Institute of Materia Medica, Shandong Academy of Medical Sciences, Jinan, Shandong 250062, PR China
School of Pharmaceutical Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
1 5 1 3 1 3
Eleven new 14,15-seco-pregnane-type steroidal glycosides, cynanosides P –P , Q –Q , R –R , and a novel
Received 29 February 2008
Received in revised form 9 October 2008
Accepted 14 October 2008
Available online 1 November 2008
1
2,13-seco-14,18-nor-pregnane-type steroidal glycoside, cynanoside S, were isolated from the roots of
Cynanchum atratum, together with four known compounds, atratoside C, sublanceoside E3, chekiangen-
soside C and cynatroside B. Their structures were determined on the basis of spectroscopic analysis and
chemical evidence.
©
2008 Elsevier Inc. All rights reserved.
Keywords:
Steroidal glycosides
Pregnane glycosides
Cynanosides
Cynanchum atratum
Asclepiadaceae
1
. Introduction
and cynatroside B [9]. The structures of the new compounds
were established by extensive spectroscopic analysis including
1D and 2D NMR experiment as well as circular dichroic method.
Cynanosides P –P5, Q –Q , R –R (1–11) and the four known
C-21 steroidal glycosides are natural hormone precursors that
act as the bioactive constituents of many medical plants and exhibit
various bioactivities. This class of compounds contains a preg-
nane skeleton. The plant, Cynanchum sp. (Asclepiadaceae), has
been reported to be rich in C-21 steroidal glycosides. Their struc-
tures were classified into polyhydroxypregnane-type glycosides
1
1
3
1
3
compounds belong to the 14,15-seco-pregnane-type steroidal gly-
cosides. Among them, cynanosides P –P5 (1–5) were characterized
1
by the presence of a butenolide moiety at C-13 of the agly-
cone, which we report for the first time as the pregnane type
glycosides. Cynanosides S (12), a new secopregnane glycoside,
was classified as a 12,13-seco-14,18-nor-pregnane-type glycoside
(Fig. 1).
[
1,2] and seco-pregnane-type glycosides. The seco-pregnane-type
glycosides were further classified into 14,15-seco-pregnane-type
3,4] and 13,14:14,15-diseco-pregnane-type glycosides [5,6]. The
[
roots of Cynanchum atratum have been used in traditional Chinese
medicine for the treatment of urinary tract infection, gonorrhea,
edema and rheumatic arthralgia [7]. Previous chemical stud-
ies had reported the occurrences of pregnane glycosides in the
roots of C. atratum, including sixteen 14,15-seco-pregnane-type
glycosides [8–10] and twenty 13,14:14,15-diseco-pregnane-type
glycosides [9,11–14]. As a result of our on-going studies of the
chemical constituents of the roots of C. atratum, herein we report
the isolation and structural determination of twelve new preg-
2
. Experimental
2.1. General
The spectrometers for IR, UV, CD, NMR, FABMS, HRFABMS and
optical rotations, as well as the separation instruments and mate-
rials were the same as in our previous report [14].
nane glycosides, cynanosides P –P5 (1–5), Q –Q (6–8), R –R3
1
1
3
1
2.2. Plant material
(
9–11) and cynanoside S (12), along with four known compounds,
atratoside C [8], sublanceoside E3 [15], chekiangensoside C [16]
Plant material used in this research was collected from Hebei
province of PR China in November 2003 and identified as C. atra-
tum Bge. by Prof. Qishi Sun (Shenyang Pharmaceutical University).
Voucher specimens are deposited in the Faculty of Pharmaceutical
Sciences, Toho University.
∗ Corresponding author. Tel.: +81 47 472 1161; fax: +81 47 472 1404.
E-mail address: liwei@phar.toho-u.ac.jp (W. Li).
0
039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.10.007

H. Bai et al. / Steroids 74 (2009) 198–207
199
C (3.00 g) was separated by Sephadex LH-20 CC with MeOH to give
three fractions (C –C ). The major fraction C (1.77 g) was further
1
3
2
purified by HPLC (50% CH CN) to give 1 (27 mg), 3 (16 mg) and
3
cynatroside B (138 mg). Fraction D (7.04 g) was chromatographed
on Sephadex LH-20 CC with MeOH to give three fractions (D –D ).
1
3
Fraction D2 (4.67 g) was fractionated by HPLC (50% CH CN) to give
3
7
(14 mg), 12 (5 mg) and sublanceoside E3 (138 mg). Further sepa-
ration of fraction 3 (19.1 g) was achieved by ODS CC with 50%, 80%
and 100% MeOH. The fraction eluted with 80% MeOH (7.90 g) was
purified by HPLC (45% CH CN) to give 4 (4 mg), 8 (14 mg), 11 (57 mg)
3
and atratoside C (128 mg). In addition, the H O fraction was evap-
2
◦
orated under reduced pressure at 40 C to remove EtOAc, and then
subjected to a Diaion HP-20 column with 30%, 50% and 100% MeOH.
The 100% MeOH fraction (21.0 g) was then chromatographed on a
silica gel column using CHCl /MeOH/H O (60:10:0.5, 60:20:3 and
3
2
6
:4:1) to give five fractions (I–IV). Fraction II (2.82 g) was subjected
to ODS CC with 30%, 60% and 90% MeOH. The fraction eluted with
0% MeOH (0.63 g) was purified by HPLC (75% MeOH) to give 2
6
(
(
14 mg) and 5 (4 mg). Further separation of the 90% MeOH fraction
1.11 g) was achieved by HPLC with 45% CH CN to give 6 (34 mg)
3
and chekiangensoside C (43 mg).
2
.3.1. Cynanoside P1 (1)
Amorphous powder, [˛]D −97.1 (c 1.10, CHCl , 24 C). UV
◦
◦
3
−
4
(
ꢁ
MeOH) ꢀmax nm (log ε): 206.5 (4.10). CD (c 1.27 × 10 M, MeOH),
ε (nm): −2.60 (296.0), −6.22 (224.5), +8.12 (202.5). IR (KBr)
−
1
ꢂmax: 3418, 2931, 1712, 1652, 1070 cm . FABMS (positive) m/z:
8
+
15 [M+Na] . HRFABMS (positive): observed 815.4210, calcd for
+
1
C
42H64O14 Na [M+Na] , 815.4194. H NMR (500 MHz, C5D5N) and
C NMR (125 MHz, C5D5N): see Tables 1 and 2.
1
3
2
.3.2. Cynanoside P2 (2)
Amorphous powder, [˛]D −84.9 (c 0.94, CHCl , 24 C), UV
◦
◦
3
−
4
(
ꢁ
MeOH) ꢀmax nm (log ε): 205.0 (4.15). CD (c 1.06 × 10 M, MeOH),
ε (nm): −3.11 (296.5), −7.07 (224.5), +12.98 (202.0). IR (KBr)
−
1
ꢂmax: 3419, 2931, 1748, 1623, 1092 cm . FABMS (positive) m/z:
9
+
77 [M+Na] . HRFABMS (positive): observed 977.4742, calcd for
+
1
C
48H74 O19 Na [M+Na] , 977.4722. H NMR (500 MHz, C5D5N) and
C NMR (125 MHz, C5D5N): see Tables 1 and 2.
1
3
2
.3.3. Cynanoside P3 (3)
Amorphous powder, [˛]D −65.9 (c 1.19, CHCl , 24 C), UV
◦
◦
3
−
4
(
ꢁ
MeOH) ꢀmax nm (log ε): 205.0 (4.06). CD (c 1.26 × 10 M, MeOH),
ε (nm): −1.29 (292.0), +0.38 (246.0), −6.45 (205.0). IR (KBr)
−
1
ꢂmax: 3427, 2930, 1752, 1652, 1096 cm . FABMS (positive) m/z:
8
+
15 [M+Na] . HRFABMS (positive): observed 815.4208, calcd for
+
1
C
42H64O14 Na [M+Na] , 815.4193. H NMR (500 MHz, C5D5N) and
C NMR (125 MHz, C5D5N): see Tables 1 and 2.
1
3
Fig. 1. Structures of 1–12.
2
.3.4. Cynanoside P4 (4)
Amorphous powder, [˛]D −62.7 (c 0.27, CHCl , 24 C), UV
◦
◦
3
−
4
(
ꢁ
MeOH) ꢀmax nm (log ε): 203.5 (3.98). CD (c 1.07 × 10 M, MeOH),
2.3. Extraction and isolation
ε (nm): −1.42 (293.5), +0.84 (244.0), −7.18 (209.5). IR (KBr)
−
1
ꢂmax: 3427, 2928, 1747, 1652, 1093 cm . FABMS (positive) m/z:
9
The roots of C. atratum (1.8 kg) were extracted with MeOH, and
+
77 [M+Na] . HRFABMS (positive): observed 977.4755, calcd for
then partitioned between EtOAc and H O by the same procedure as
2
+
1
C
48H74 O Na [M+Na] , 977.4722. H NMR (500 MHz, C5D5N) and
in our previous report [14]. The EtOAc extract (162 g) was subjected
19
1
3
C NMR (125 MHz, C5D5N): see Tables 1 and 2.
to silica gel CC with a gradient of CHCl /MeOH to give four fractions
3
(
1–4). Fraction 2 (82.9 g) was loaded on a Diaion HP-20 column and
eluted with 50%, 70%, 90% and 100% MeOH. The 90% MeOH fraction
25.6 g) was separated by chromatography on a silica gel column
using CHCl /MeOH (99:1, 97:3, 95:5 and 9:1) to give five fractions
2.3.5. Cynanoside P5 (5)
◦
◦
(
Amorphous powder, [˛]D −71.3 (c 0.26, CHCl , 24 C), UV
3
−
5
(MeOH) ꢀmax nm (log ε): 203.5 (4.02). CD (c 9.26 × 10 M, MeOH),
ꢁε (nm): −2.57 (295.0), +2.37 (254.0), −3.81 (230.0), +6.90 (208.5).
3
(
A–E). Fraction B (6.17 g) was subjected to Sephadex LH-20 CC with
−
1
MeOH to give three fractions (B –B ). Fraction B (1.72 g) was puri-
IR (KBr) ꢂmax: 3426, 2925, 1739, 1652, 1096 cm . FABMS (positive)
1
3
2
+
fied by HPLC (50% CH CN) to give 9 (50 mg) and 10 (12 mg). Fraction
m/z: 1007 [M+Na] . HRFABMS (positive): observed 1007.4824, calcd
3

2
00
H. Bai et al. / Steroids 74 (2009) 198–207
Table 1
1
3 C NMR data (125 MHz) for 1–11 in C5D5N.
Position
1
2
3
4
5
6
7
8
9
10
83.5
207.0
77.0
39.7
134.4
127.0
27.2
11
83.6
207.0
77.0
39.7
134.4
127.0
27.2
43.0
50.8
48.8
23.0
38.6
47.9
212.0
139.4
111.5
123.5
24.0
14.0
1
2
3
4
5
6
7
8
9
45.3
70.0
85.1
37.6
45.3
70.0
85.1
37.6
45.3
70.0
85.1
37.6
45.3
70.0
85.1
37.6
45.3
70.0
85.2
37.6
45.4
70.0
85.1
37.5
45.4
45.4
70.0
85.1
52.0
70.0
85.2
37.5
205.6
78.5
39.4
136.9
123.5
27.0
37.5
139.1
121.3
26.6
41.9
139.1
121.3
26.6
41.9
139.1
121.3
26.4
41.7
139.1
121.3
26.4
41.7
139.2
121.3
26.9
41.9
138.9
121.2
25.8
138.9
121.2
25.8
43.0
52.4
38.9
22.4
39.8
76.3
213.1
140.9
110.5
121.4
138.9
121.2
25.8
43.0
52.4
38.9
22.4
39.8
76.3
213.1
140.9
110.5
121.4
43.0
52.4
38.9
22.4
39.8
76.3
41.7
43.1
50.5
38.9
19.9
35.3
51.0
50.5
38.9
19.8
35.3
51.0
50.9
38.9
20.2
37.6
50.9
38.9
20.2
37.6
50.2
38.9
19.7
33.5
51.6
50.0
43.3
20.3
38.2
47.6
50.8
48.9
23.0
38.6
47.9
211.9
139.3
111.5
123.5
24.0
14.0
1
0
1
1
1
2
1
3
51.1
51.1
1
4
210.9
172.4
118.8
176.4
23.0
19.9
210.8
172.4
118.8
176.4
23.0
19.8
211.8
172.5
118.1
177.2
22.2
19.8
80.5
19.8
211.8
172.5
118.1
177.2
22.2
19.8
80.5
19.8
210.6
169.2
123.5
170.1
22.6
19.8
110.1
24.8
50.6
213.1
140.9
110.5
121.4
211.8
139.4
111.4
123.5
23.9
19.7
1
5
1
6
1
7
1
8
1
9
19.8
19.9
149.9
12.8
19.8
149.9
12.8
2
0
80.6
20.1
80.6
20.1
149.9
12.8
148.2
14.7
148.2
14.7
148.2
14.7
2
1
2
2
␤
-d-cym I
␤-d-cym I
97.5
35.2
77.5
82.1
69.6
18.4
57.4
␤-d-cym I
97.5
35.3
77.5
82.1
69.6
18.4
57.4
␤-d-cym I
97.5
35.3
77.5
82.1
69.6
18.4
57.4
␤-d-cym I
97.5
35.3
77.5
82.1
69.6
18.4
57.4
␤-d-cym I
97.4
35.2
77.5
82.1
69.6
18.3
57.4
␤-d-cym
97.8
37.1
77.9
83.0
69.4
18.2
59.0
␤-d-cym
97.8
37.1
77.9
83.0
69.4
18.2
59.0
␤-d-cym I
95.6
34.7
77.2
81.8
69.7
18.6
57.2
␤-d-cym I
95.9
34.7
77.2
81.8
69.7
18.6
57.3
␤-d-cym I
95.9
34.6
77.2
81.8
69.7
18.6
57.2
1
2
3
4
5
6
3
97.5
35.2
77.5
82.1
69.6
18.4
57.4
-OCH3
-OCH3
-OCH3
␣
-l-dgn
␣-l-dgn
101.1
32.4
74.6
73.6
67.6
17.9
55.4
␣-l-dgn
101.1
32.5
74.6
73.9
67.6
17.9
55.4
␣-l-dgn
101.1
32.4
74.6
73.6
67.6
17.9
55.4
␣-l-dgn
101.1
32.4
74.6
73.6
67.6
17.9
55.4
␣-l-dgn
101.1
32.4
74.6
73.6
67.6
17.9
55.4
␤-d-dgt
100.6
39.8
67.7
82.3
␤-d-dgt
100.5
39.8
67.5
82.2
␣-l-dgn
100.8
32.5
74.7
73.9
67.6
17.9
55.4
␣-l-dgn
100.8
32.5
74.7
74.0
67.7
17.9
55.4
␣-l-dgn
100.8
32.5
74.6
73.7
67.6
17.9
55.4
1
2
3
4
5
6
3
101.1
32.5
74.6
73.9
67.6
17.9
55.4
68.8
18.6
68.7
18.6
␤
-d-cym II
␤-d-cym II
99.3
36.2
78.2
83.0
69.6
18.5
58.6
␤-d-cym II
99.5
35.4
79.0
74.2
71.1
18.9
58.0
␤-d-cym II
99.3
36.2
78.2
83.0
69.6
18.5
58.6
␤-d-cym II
99.3
36.2
78.2
83.0
69.6
18.5
58.6
␤-d-cym II
99.3
36.2
78.1
83.0
69.6
18.5
58.6
␣-l-ole
100.4
35.8
78.9
76.9
69.6
18.5
57.1
␣-l-ole
99.9
35.5
78.8
82.2
68.1
␤-d-cym II
99.5
35.4
78.9
74.2
71.1
18.8
58.0
␤-d-cym II
99.5
35.4
79.0
74.2
71.2
18.8
58.0
␤-d-cym II
99.3
36.2
78.2
83.0
69.6
18.5
58.6
1
2
3
4
5
6
3
99.5
35.4
78.9
74.2
71.1
18.8
58.0
18.5
56.8
␤
-d-glc
␤-d-glc
106.5
75.5
78.4
71.9
␤-d-glc
106.5
75.5
78.4
71.9
␤-d-glc
106.5
75.4
78.4
71.9
␤-d-glc
105.1
76.0
78.4
72.0
␤-d-glc
106.5
75.5
78.4
71.9
1
2
3
4
5
6
106.5
75.5
78.4
71.9
78.4
63.0
78.4
63.0
78.4
63.0
78.4
63.0
78.4
63.1
78.4
63.0
cym: cymaropyranosyl; dgn: diginopyranosyl; dgt: digitoxopyranosyl; ole: oleandropyranosyl; glc: glucopyranosyl.
for C 9H76O20Na [M+Na] , 1007.4827. 1H NMR (500 MHz, C5D5N)
+
+
787 [M+Na] . HRFABMS (positive): observed 787.3879, calcd for
4
1
3
+
1
and C NMR (125 MHz, C5D5N): see Tables 1 and 2.
C40H60O14 Na [M+Na] , 787.3881. H NMR (500 MHz, C5D5N) and
C NMR (125 MHz, C5D5N): see Tables 1 and 3.
13
2
.3.6. Cynanoside Q1 (6)
Amorphous powder, [˛]D −71.0 (c 0.91, CHCl , 24 C), IR (KBr)
2
.3.8. Cynanoside Q3 (8)
Amorphous powder, [˛]D −45.6 (c 0.99, CHCl , 24 C), IR (KBr)
◦
◦
3
◦
◦
−1
3
ꢂmax: 3425, 2930, 1704, 1652, 1077 cm . FABMS (positive) m/z:
9
−
1
ꢂmax: 3420, 2931, 1707, 1652, 1057 cm . FABMS (positive) m/z:
9
+
63 [M+Na] . HRFABMS (positive): observed 963.4571, calcd for
+
49 [M+Na] . HRFABMS (positive): observed 949.4438, calcd for
+
1
C
7H72O Na [M+Na] , 963.4566. H NMR (500 MHz, C5D5N) and
4
19
+
1
C
46H70O Na [M+Na] , 949.4409. H NMR (500 MHz, C5D5N) and
1
3
19
C NMR (125 MHz, C5D5N): see Tables 1 and 3.
1
3
C NMR (125 MHz, C5D5N): see Tables 1 and 3.
2
.3.7. Cynanoside Q2 (7)
Amorphous powder, [˛]D −57.9 (c 1.00, CHCl , 24 C), IR (KBr)
2.3.9. Cynanoside R1 (9)
◦
◦
◦
◦
Amorphous powder, [˛]D −83.5 (c 0.67, CHCl , 24 C),
3
3
−1
−1
ꢂmax: 3429, 2931, 1708, 1652, 1056 cm . FABMS (positive) m/z:
IR (KBr) ꢂmax: 3429, 2926, 1706, 1652, 1090 cm
. FABMS

H. Bai et al. / Steroids 74 (2009) 198–207
201
Table 2
1
H NMR data (500 MHz) for 1–5 in C5D5N.
Position
1
1
2
3
4
5
1.33a
.42 (dd, 12.8, 4.6)
1.33 (t, 12.4)
2.42 (dd, 13.1, 4.6)
1.31 (t, 12.3)
2.39 (dd, 13.0, 4.6)
1.32 (t, 12.9)
2.39 (dd, 12.9, 4.6)
1.34 (t, 12.4)
2.42 (dd, 13.1, 4.6)
2
2
3
4
6
7
8
9
4.06 (ddd, 12.0, 9.0, 4.6)
3.64 (m)
4.06 (ddd, 12.8, 8.9, 4.6)
3.64 (m)
4.05 (ddd, 12.0, 9.0, 4.6)
3.63 (m)
2.53
5.42 (br s)
2.25 (m)
2.72 (ddd, 12.4, 10.4, 6.2)
1.56 (td, 12.4, 4.6)
1.77 (m), 1.71 (m)
1.96 (m), 1.75 (m)
6.18 (d, 1.6)
4.05 (ddd, 11.6, 8.9, 4.6)
3.63 (m)
2.54
5.42 (br s)
2.25 (m)
2.73 (ddd, 12.2, 10.1, 6.1)
1.56 (td, 12.2, 4.4)
1.77 (m), 1.70 (m)
1.96 (m), 1.75 (m)
6.19 (d, 1.6)
4.06 (ddd, 12.2, 9.1, 4.6)
3.64 (m)
2.53 (m)
5.43 (t like, 2.5)
2.25 (m)
a
a
a
a
2.53
2.53
5.40
5.41a
a
2.25a
2.25 (m)
2.66 (ddd, 12.6, 10.2, 6.0)
1.57 (m)
1.83 (m), 1.73 (m)
1.86 (m)
6.17 (d, 1.6)
1.39 (s)
2.67 (ddd, 12.6, 10.3, 6.0)
1.57 (m)
1.83 (m), 1.73 (m)
1.86 (m)
6.17 (d, 1.6)
1.40 (s)
2.70 (ddd, 12.4, 10.7, 5.9)
a
1.59
1
1
1.72 (m)
2.32 (m), 1.85 (m)
6.41(s)
1
2
1
6
18
1.44 (s)
1.44 (s)
1.49 (s)
19
1.11 (s)
1.10 (s)
1.11 (s)
1.11 (s)
1.10 (s)
2
21
0
5.41 (qd, 6.7, 1.6)
1.37 (d, 6.7)
5.42 (qd, 6.7, 1.6)
1.36 (d, 6.7)
5.55 (qd, 6.7, 1.6)
1.39 (d, 6.7)
5.54 (qd, 6.7, 1.6)
1.39 (d, 6.7)
1.65 (s)
3.16 (s)
2
2
␤-d-cym I
␤-d-cym I
␤-d-cym I
␤-d-cym I
␤-d-cym I
1
2
5.21 (dd, 9.6, 1.8)
5.20 (dd, 9.6, 1.8)
5.21 (dd, 9.7, 1.6)
5.20 (dd, 9.6, 1.8)
5.21 (dd, 9.6, 1.9)
1.83a
1.82 (ddd, 13.8, 9.6, 2.3)
2.48 (ddd, 13.8, 3.2, 1.8)
1.83 (ddd, 13.6, 9.7, 2.1)
1.82 (ddd, 13.7, 9.6, 2.2)
1.81 (ddd, 13.5, 9.6, 2.1)
2.48 (ddd, 13.5, 3.4, 1.9)
a
a
a
2.48
2.48
2.48
3
4
5
6
3
3.93 (q like, 2.8)
3.45a
3.92 (q like, 2.8)
3.45 (dd, 9.6, 2.8)
4.25 (dq, 9.6, 6.2)
1.31 (d, 6.2)
3.93 (q like, 2.8)
3.91 (q like, 2.8)
3.45 (dd, 9.6, 2.8)
4.25
1.31 (d, 6.2)
3.55 (s)
3.92 (q like, 2.8)
3.45 (dd, 9.4, 2.8)
4.25
1.31 (d, 6.2)
3.54 (s)
a
3.45
4.25
4.25a
a
a
a
1.33 (d, 6.2)
3.56 (s)
1.32 (d, 6.2)
3.56 (s)
-OCH3
-OCH3
-OCH3
3.54 (s)
␣-l-dgn
␣-l-dgn
5.14 (br d, 2.7)
␣-l-dgn
5.16 (br d, 3.2)
␣-l-dgn
5.14 (br s)
␣-l-dgn
5.13 (br d, 2.0)
1
2
5.17 (br d, 2.4)
2.36 (td, 12.6, 2.4)
2.34 (td, 12.3, 2.7)
1.99 (dd, 12.3, 4.4)
2.35 (td, 12.4, 3.2)
2.00 (dd, 12.4, 4.4)
2.33 (td, 12.5, 3.7)
1.99 (dd, 12.5, 4.6)
2.33 (td, 12.3, 2.0)
1.99 (dd, 12.3, 4.2)
2.00 (dd, 12.6, 4.4)
3
4
5
6
3
3.83 (ddd, 12.6, 4.4, 2.7)
4.09 (br s)
3.80 (ddd, 12.3, 4.4, 3.0)
4.04 (br s)
3.83 (ddd, 12.4, 4.4, 2.7)
4.09 (br s)
3.80 (ddd, 12.5, 4.6, 2.9)
4.05 (br d, 2.9)
3.80 (ddd, 12.3, 4.2, 3.0)
4.04 (br s)
4.25a
1.47 (d, 6.6)
3.45 (s)
4.22
a
4.25
a
4.22
a
4.22
a
1.42 (d, 6.4)
3.40 (s)
1.47 (d, 6.6)
3.46 (s)
1.42 (d, 6.4)
3.41 (s)
1.42 (d, 6.4)
3.40 (s)
␤-d-cym II
␤-d-cym II
5.14 (dd, 9.6, 1.6)
␤-d-cym II
5.13 (dd, 9.6, 1.6)
␤-d-cym II
5.15 (dd, 9.8, 1.8)
␤-d-cym II
5.14 (dd, 9.6, 2.0)
1
2
5.13 (dd, 9.6, 1.6)
1.86a
1.86 (ddd, 13.7, 9.6, 2.5)
2.38 (ddd, 13.7, 3.5, 1.6)
1.86 (ddd, 13.3, 9.6, 2.3)
1.87 (ddd, 13.5, 9.8, 2.5)
1.86a
2.37 (m)
a
a
a
2.49
2.49
2.38
3
4
5
6
3
3.74 (q like, 2.8)
3.57 (dd, 9.5, 2.8)
4.12 (dq, 9.5, 6.2)
1.54 (d, 6.2)
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)
3.74 (q like, 3.0)
3.58 (dd, 9.5, 3.0)
4.12 (dq, 9.5, 6.2)
1.54 (d, 6.2)
4.10 (q like, 2.9)
3.71 (dd, 9.8, 2.9)
4.29 (dq, 9.8, 6.2)
1.65 (d, 6.2)
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)
3.47 (s)
3.52 (s)
3.47 (s)
3.52 (s)
3.52 (s)
␤-d-glc
␤-d-glc
␤-d-glc
1
2
3
4
5
4.95 (d, 7.8)
4.01 (dd, 9.6, 7.8)
4.25 (t, 9.6)
4.20 (t, 9.6)
3.97 (ddd, 9.6, 5.6, 2.7)
4.94(d, 7.8)
4.02 (dd, 8.7, 7.8)
4.24 (t, 8.7)
4.21 (dd, 9.2, 8.7)
3.97 (ddd, 9.2, 5.2, 2.5)
4.94 (d, 7.7)
4.01 (dd, 8.7, 7.7)
4.25 (t, 8.7)
4.21 (t, 8.7)
3.97 (ddd, 8.7, 5.3, 2.4)
6
4.41 (dd, 11.2, 5.6)
4.41 (dd, 11.7, 5.2)
4.56 (dd, 11.7, 2.5)
4.41 (dd, 11.7, 5.3)
4.56 (dd, 11.7, 2.4)
4.56 (dd, 11.2, 2.7)
cym: cymaropyranosyl; dgn: diginopyranosyl; glc: glucopyranosyl.
a
Overlapped signals.
C
42H62O14 Na [M+Na] , 813.4037. 1H NMR (500 MHz, C D N) and
+
5
5
(
7
(
positive) m/z: 797 [M+Na]+. HRFABMS (positive): observed
+
1
13
97.4117, calcd for C42H62
O
13
Na [M+Na] , 797.4088. H NMR
5 5
C NMR (125 MHz, C D N): see Tables 1 and 4.
500 MHz, C5D5N) and 3C NMR (125 MHz, C5D5N): see
1
Tables 1 and 4.
2.3.11. Cynanoside R3 (11)
◦
◦
Amorphous powder, [˛]D −66.8 (c 1.09, CHCl , 24 C), IR (KBr)
3
−
1
2
.3.10. Cynanoside R2 (10)
Amorphous powder, [˛]D −69.8 (c 0.99, CHCl , 24 C), IR (KBr)
ꢂmax: 3427, 2926, 1704, 1652, 1076 cm . FABMS (positive) m/z:
◦
◦
+
975 [M+Na] . HRFABMS (positive): observed 975.4541, calcd for
3
−1
+
1
ꢂmax: 3427, 2926, 1704, 1652, 1090 cm . FABMS (positive) m/z:
8
C48H72O19 Na [M+Na] , 975.4565. H NMR (500 MHz, C5D5N) and
+
13
13 [M+Na] . HRFABMS (positive): observed 813.4062, calcd for
C NMR (125 MHz, C5D5N): see Tables 1 and 4.

2
02
H. Bai et al. / Steroids 74 (2009) 198–207
Table 3
1
H NMR data (500 MHz) for 6–8 in C5D5N.
Position
6
7
8
1
1.29 (t, 12.8)
.35 (dd, 12.8, 4.3)
1.28 (t, 12.6)
2.33 (dd, 12.6, 4.8)
1.28 (t, 12.6)
2.33 (dd, 12.6, 4.6)
2
2
3
3.99a
3.96 (ddd, 12.6, 9.0, 4.8)
3.57 (ddd, 11.8, 9.0, 5.5)
3.99a
3.59 (ddd, 11.4, 8.9, 5.7)
3.58 (ddd, 11.5, 8.9, 5.6)
4
2.50 (dd, 13.9, 5.7)
2.51 (dd, 13.1, 5.5)
2.45
2.48 (dd, 14.0, 5.6)
2.45
a
a
a
2.46
6
7
8
9
5.43 (dd, 3.2, 1.7)
2.65 (m), 2.22 (m)
2.70 (m)
1.60 (td, 11.4, 5.5)
1.78 (m), 1.74 (m)
5.43 (br d, 4.8)
2.65 (m), 2.21 (m)
2.69 (m)
1.59 (td, 11.3, 5.3)
1.78 (m), 1.73 (m)
5.43 (br d, 4.9)
2.65 (m), 2.21 (m)
2.69 (m)
1.58 (td, 11.3, 5.4)
1.78 (m), 1.74 (m)
11
12
1.97 (td, 13.5, 4.5)
1.97a
1.97a
2.89 (dt, 13.5, 3.2)
2.88 (dt, 13.3, 3.1)
2.89 (dt, 13.5, 3.2)
1
5
7.53 (d, 2.0)
6.69 (d, 2.0)
0.88 (s)
7.52 (d, 2.0)
6.68 (d, 2.0)
0.87 (s)
7.53 (d, 1.9)
6.69 (d, 1.9)
0.87 (s)
1
6
19
21
2.20 (s)
2.19 (s)
2.20 (s)
␤-d-cym I
␤-d-cym
␤-d-cym
1
2
5.18 (dd, 9.6, 1.8)
5.20 (dd, 9.6, 1.9)
5.20 (dd, 9.6, 1.8)
1.82 (ddd, 13.5, 9.6, 2.3)
1.90 (ddd, 13.3, 9.6, 2.3)
2.38 (ddd, 13.3, 3.7, 1.9)
1.90 (ddd, 13.5, 9.6, 2.3)
a
2.46 (ddd, 13.5, 3.4, 1.8)
2.37
3
4
5
6
3
3.91 (q like, 2.8)
3.44 (dd, 9.6, 2.8)
4.24 (dq, 9.6, 6.2)
1.30 (d, 6.2)
4.11 (q like, 2.8)
3.49 (dd, 9.6, 2.8)
4.22 (dq, 9.6, 6.2)
1.29 (d, 6.2)
4.10 (q like, 2.8)
3.49 (dd, 9.6, 2.8)
4.22
1.29 (d, 6.2)
3.64 (s)
a
-OCH3
-OCH3
-OCH3
3.54 (s)
3.65 (s)
␣-l-dgn
␤-d-dgt
5.35 (dd, 9.5, 1.7)
␤-d-dgt
5.36 (dd, 9.6, 1.5)
1
2
5.13 (br d, 3.4)
2.33 (td, 12.3, 3.4)
1.93a
1.93a
a
a
1.99 (dd, 12.3, 4.4)
2.40
2.40
3
4
5
6
3
3.79 (ddd, 12.3, 4.4, 3.0)
4.04 (br d, 3.0)
4.60 (ddd, 3.5, 3.2, 2.6)
3.53 (dd, 9.4, 2.6)
4.40 (dq, 9.4, 6.2)
1.45 (d, 6.2)
4.53a
3.49 (dd, 9.6, 2.7)
a
a
4.22
4.36
1.41 (d, 6.6)
3.41 (s)
1.42 (d, 6.2)
␤-d-cym II
␣-l-ole
5.26 (d, 3.2)
␣-l-ole
5.21(br s)
1
2
5.13 (dd, 9.6, 1.6)
1.86 (ddd, 13.3, 9.6, 2.5)
1.74 (m)
1.69 (m)
a
a
2.37 (ddd, 13.3, 3.4, 1.6)
2.47
2.44
3
4
5
6
3
4.09 (q like, 2.8)
3.70 (dd, 9.6, 2.8)
4.28 (dq, 9.6, 6.2)
1.64 (d, 6.2)
3.83 (ddd, 11.4, 9.2, 5.0)
3.54 (t, 9.2)
4.41 (dq, 9.2, 6.2)
1.52 (d, 6.2)
4.00a
3.82 (t, 9.1)
4.37 (dq, 9.1, 6.2)
1.55 (d, 6.2)
3.35 (s)
3.52 (s)
3.35 (s)
␤-d-glc
␤-d-glc
1
2
3
4
5
4.93 (d, 7.8)
5.27 (d, 7.8)
3.98 (dd, 8.4, 7.8)
4.24 (t, 8.4)
4.21 (dd, 9.1, 8.4)
3.92 (ddd, 9.1, 5.3, 2.5)
4.00 (dd, 8.3, 7.8)
4.24 (dd, 8.9, 8.3)
4.20 (t, 8.9)
3.96 (ddd, 8.9, 5.3, 2.3)
6
4.40 (dd, 11.7, 5.3)
4.35a
4.53
a
4.55 (dd, 11.6, 2.3)
cym: cymaropyranosyl; dgn: diginopyranosyl; dgt: digitoxopyranosyl; ole: oleandropyranosyl; glc: glucopyranosyl.
a
Overlapped signals.
2
.3.12. Cynanoside S (12)
Amorphous powder, [˛]D −63.4 (c 0.43, CHCl , 24 C), IR (KBr)
2.4. Acid hydrolysis of 1, 3, 5, 6, 7, 9, 10 and 12
◦
◦
3
−1
ꢂmax: 3427, 2928, 1714, 1632, 1093 cm . FABMS (positive) m/z:
8
Acid hydrolysis of 1, 3, 5, 6, 7, 9, 10 and 12 (each 1 mg) were
carried out by the same procedure as in our previous report [14].
Each aqueous layer was neutralized by passing through an ion-
exchange resin (Amberlite MB-3, Organo, Tokyo, Japan) column
+
19 [M+Na] . HRFABMS (positive): observed 819.3787, calcd for
+
1
C
40H60O16 Na [M+Na] , 819.3779. H NMR (500 MHz, C5D5N) and
C NMR (125 MHz, C5D5N): see Table 5.
1
3

H. Bai et al. / Steroids 74 (2009) 198–207
203
Table 4
1
H NMR data (500 MHz) for 9–11 in C5D5N.
Position
9
10
11
4.39 (d, 1.3)
1
2.30 (d, 12.6)
.64 (d, 12.6)
4.36 (br s)
2
3
4
4.79 (dd, 12.5, 7.3)
4.85 (ddd, 12.6, 7.6, 1.3)
4.87 (ddd, 12.6, 7.5, 1.3)
3.00 (dd, 13.5, 7.5)
2.98 (dd, 13.7, 7.3)
2.99 (dd, 13.5, 7.6)
a
a
a
2.70
2.72
2.75
6
7
8
9
5.56 (br s)
2.37 (m)
2.69
1.74 (m)
1.73 (m), 1.57 (m)
1.95 (m)
7.46 (d, 1.8)
6.42 (d, 1.8)
1.49 (s)
5.76 (br d, 5.5)
2.40 (m)
5.76 (br d, 5.7)
2.40 (m)
a
2.68 (ddd, 12.3, 10.5, 5.3)
2.14 (td, 11.7, 4.1)
2.88 (m), 1.96 (m)
2.08 (m), 1.99 (m)
7.42 (d, 1.8)
2.67 (ddd, 12.6, 10.3, 5.6)
2.15 (td, 11.9, 3.8)
2.91 (m), 1.96 (m)
2.08 (m), 1.99 (m)
7.44 (d, 1.8)
1
1
1
2
1
5
1
6
6.40 (d, 1.8)
1.51 (s)
6.42 (d, 1.8)
1.52 (s)
18
19
1.08 (s)
1.15 (s)
21
2.24 (s)
2.24 (s)
2.24 (s)
␤-d-cym I
␤-d-cym I
␤-d-cym I
1
2
5.32 (dd, 9.2, 1.9)
5.32 (dd, 9.2, 2.1)
5.32 (dd, 9.2, 1.8)
1.93 (ddd, 13.8, 9.2, 2.1)
1.90 (ddd, 13.8, 9.2, 2.2)
1.92a
a
2.52 (ddd, 13.8, 3.9, 1.9)
2.49
2.50 (ddd, 14.0, 4.1, 1.8)
3
4
5
6
3
3.95 (q like, 2.8)
3.52 (dd, 9.2, 2.8)
4.23 (dq, 9.2, 6.4)
1.34 (d, 6.4)
3.93 (q like, 2.8)
3.50 (dd, 9.2, 2.8)
4.20 (dq, 9.2, 6.4)
1.29 (d, 6.4)
3.92 (q like, 2.8)
3.48 (dd, 9.6, 2.8)
4.22
1.29 (d, 6.2)
3.52 (s)
a
-OCH3
-OCH3
-OCH3
3.53 (s)
3.52 (s)
␣-l-dgn
␣-l-dgn
5.14 (br d, 3.4)
␣-l-dgn
5.14 (br s)
1
2
5.14 (br d, 3.4)
2.35 (td, 12.4, 3.4)
2.34 (td, 12.3, 3.4)
2.02 (dd, 12.3, 4.5)
2.33 (td, 12.4, 3.7)
1.99 (dd, 12.4, 4.3)
2.02 (dd, 12.4, 4.4)
3
4
5
6
3
3.84 (ddd, 12.4, 4.4, 2.9)
4.08 (br d, 2.3)
4.26 (q, 6.4)
1.46 (d, 6.4)
3.45 (s)
3.82 (ddd, 12.3, 4.5, 2.8)
4.08 (br s)
4.26 (q, 6.4)
1.44 (d, 6.4)
3.81 (ddd, 12.4, 4.3, 2.8)
4.04 (br d, 2.0)
a
4.23
1.41 (d, 6.4)
3.41 (s)
3.44 (s)
␤-d-cym II
␤-d-cym II
5.12 (dd, 9.6, 1.8)
␤-d-cym II
5.14 (dd, 9.6, 1.8)
1
2
5.12 (dd, 9.6, 1.5)
1.86 (ddd, 13.8, 9.6, 2.3)
1.84 (ddd, 13.8, 9.6, 2.5)
1.87 (ddd, 13.4, 9.6, 2.3)
a
a
2.49 (m)
2.47
2.38
3
4
5
6
3
3.74 (q like, 2.9)
3.56 (dd, 9.6, 2.9)
4.12 (dq, 9.6, 6.2)
1.54 (d, 6.2)
3.73 (q like,2.9)
3.53 (dd, 9.8, 2.9)
4.09 (dq, 9.8, 6.4)
1.52 (d, 6.4)
4.10 (q like, 2.8)
3.70 (dd, 9.6, 2.8)
4.28 (dq, 9.6, 6.2)
1.64 (d, 6.2)
3.47 (s)
3.46 (s)
3.52 (s)
␤
-d-glc
1
2
3
4
5
4.93 (d, 7.8)
4.01 (dd, 9.0, 7.8)
4.23 (t, 9.0)
4.20 (t, 9.0)
3.96 (ddd, 9.0, 5.3, 2.7)
6
4.40 (dd, 11.7, 5.3)
4.56 (dd, 11.7, 2.7)
cym: cymaropyranosyl; dgn: diginopyranosyl; glc: glucopyranosyl.
a
Overlapped signals.
and concentrated under reduced pressure to give a sugar frac-
tion.
derivatives by comparison with authentic sample. HPLC condi-
tion: column, YWC-Pack ODS-A, 150 mm × 4.6 mm i.d.; flow rate,
◦
0
.8 mL/min; column temperature, 40 C; UV wavelength, 210 nm;
2.5. Enzymatic hydrolysis of 2, 4, 8 and 11
tR, 1 7.42 min (45% CH3CN as eluate solvent), 3 5.95 min (50%
CH CN as eluate solvent), 7 5.20 min (50% CH CN as eluate sol-
3
3
Enzymatic hydrolysis of 2, 4, 8 and 11 (each 1 mg) were carried
out by the same procedure as in our previous report [14]. The MeOH
eluate was analyzed by HPLC and TLC to identify the deglucosyl
vent), 10 4.62 min (60% CH3CN as eluate solvent). TLC condition:
CHCl3/MeOH (95:5), Rf, 1 0.37; 7 0.23; 10 0.52; CHCl3/MeOH (9:1),
Rf, 3 0.65.

2
04
H. Bai et al. / Steroids 74 (2009) 198–207
Table 5
1
H (500 HMz) and 13 C NMR data (125 MHz) for 12 in C5D5N.
Position
C
H
Position
C
H
1
2
3
4
5
6
7
8
9
45.2
69.6
85.1
1.62 (t, 12.4), 2.57a
␤-d-cym I
1
2
4.04 (ddd, 11.7, 8.8, 4.6)
3.62 (ddd, 11.5, 8.8, 5.5)
97.5
35.2
5.18 (dd, 9.7, 1.9)
1.82 (ddd, 13.7, 9.7, 2.1)
a
a
37.2
2.50
2.48
140.2
119.5
32.9
71.4
3
4
5
6
77.5
82.1
69.6
18.4
57.4
3.93 (q like, 2.8)
a
5.27 (br d, 5.2)
3.46
4.26
a
2.66 (m), 2.27 (m)
5.43 (ddd, 11.4, 9.6, 5.7)
2.82 (m)
1.32 (d, 6.2)
3.57 (s)
47.5
3-OCH3
1
0
40.2
33.3
175.7
164.0
140.8
115.6
114.3
20.0
159.6
13.7
␣-l-dgn
1
2
1
1
2.78 (m), 2.70 (m)
101.1
32.5
5.17 (br d, 3.5)
1
2
2.36 (td, 12.4, 3.5)
2.00 (dd, 12.4, 4.4)
3.83 (ddd, 12.4, 4.4, 3.0)
4.10 (br s)
1
3
1
5
7.24 (d, 2.1)
6.99 (d, 2.1)
3
4
5
6
74.6
73.9
67.6
17.9
55.4
1
6
a
1
7
4.26
1
9
1.04 (s)
2.57 (s)
1.47 (d, 6.6)
3.45 (s)
2
21
0
3-OCH3
␤-d-cym II
1
2
99.5
35.4
5.13 (dd, 9.7, 1.9)
1.86 (ddd, 13.8, 9.7, 2.3)
a
2.51
3
4
5
6
79.0
74.2
71.1
18.9
58.0
3.74 (q like, 2.9)
3.58 (dd, 9.6, 2.9)
4.12 (dq, 9.6, 6.2)
1.55 (d, 6.2)
3
-OCH3
3.47 (s)
cym: cymaropyranosyl; dgn: diginopyranosyl.
a
Overlapped signals.
2.6. Determination of the absolute configuration of the
5.14 (1H, overlapped, dgn-H-1), 5.11 (1H, dd, J = 9.6, 1.6 Hz, cym II-
monosaccharides
H-1), 4.38 (1H, m, H-8), 4.21–4.25 (2H, m, cym I-H-5 and dgn-H-5),
4
.10 (1H, dq, J = 9.6, 6.2 Hz, cym II-H-5), 4.07 (1H, br.d, J = 2.1 Hz, dgn-
Identification of strophanthobiose, d-cymarose, l-dignose, d-
digitoxose and d-glucose present in the sugar fraction was carried
out by a comparison of their retention time and polarity with those
of authentic samples by the same procedure as in our previous
report [10]. tR (min): 6.55 (strophanthobiose, positive polarity),
H-4), 4.02 (1H, m, H-2), 3.90 (1H, q like, J = 3.2 Hz, cym I-H-3), 3.80
(1H, ddd, J = 12.5, 4.3, 3.5 Hz, dgn-H-3), 3.72 (1H, q like, J = 3.2 Hz,
cym II-H-3), 3.62 (1H, m, H-3), 3.60 (3H, s, C-12-OCH ), 3.58 (1H,
3
cym II-H-4), 3.53 (3H, s, cym I-OCH ), 3.45 (3H, s, cym II-OCH ),
3
3
3.44 (1H, m, cym I-H-4), 3.43 (3H, s, dgn-OCH ), 3.03 (1H, m, H-11),
3
8
.00 (d-glucose, positive polarity), 9.19 (d-cymarose, positive polar-
2.80 (1H, m, H-11), 2.43–2.59 (8H, H-1␤, H-4, H-7, H-9, cym I-H-2e
and cym II-H-2e), 2.33 (1H, td, J = 12.5, 3.5 Hz, dgn-H-2a), 2.00 (1H,
dd, J = 12.5, 4.3 Hz, dgn-H-2e), 1.84 (1H, ddd, J = 13.9, 9.6, 2.6 Hz, cym
II-H-2a), 1.80 (1H, ddd, J = 13.8, 9.6, 2.3 Hz, cym I-H-2a), 1.64 (1H,
ity), 9.46 (l-diginose, negative polarity) and 9.55 (d-digitoxose,
positive polarity). Strophanthobiose was detected for 5 and 6. d-
Cymarose and l-diginose were detected for 1, 3, 5, 6, 9, 10 and 12.
d-Cymarose and d-digitoxose were detected for 7. d-Glucose was
detected for 2, 4, 8 and 11.
t, J = 12.5 Hz, H-1␣), 1.51 (3H, d, J = 6.2 Hz, cym II-H -6), 1.44 (3H, d,
3
J = 6.6 Hz, dgn-H -6), 1.29 (3H, d, J = 6.2 Hz, cym I-H -6), 1.05 (3H, s,
3
3
H -19).
3
2.7. Alkaline hydrolysis of 12
2.8. Cytotoxic assay
1
2 (1 mg) was treated with 0.28% NaOMe in MeOH (200 ␮L) at
◦
6
0 C for 24 h. The mixture was extracted with hexane (500 ␮L).
The human HL-60 myeloid leukemia, human stomach KATO-III
The hexane extract was washed with H O (100 ␮L), and then ana-
lyzed by GC–MS under the following conditions: capillary column,
adenocarcinoma, and human lung A549 adenocarcinoma cell line
assays were performed as in our previous report [10].
2
EQUITYTM-1 (30 m × 0.25 mm × 0.25 ␮m, Supelco); injection tem-
◦
perature, 250 C; carrier N gas. The temperature of the column
3. Results and discussion
2
◦
was initially maintained at 60 C for 5 min, then raised at rate of
◦
◦
3
C/min to 240 C, where it was maintained for 5 min. Methyl 2-
Cynanoside P1 (1) was obtained as an amorphous powder. Its
molecular formula, C42H64O14 , was established by HRFABMS (m/z
methylfuran-3-carboxylate (12b) was confirmed by comparison of
+
+
the retention time at 8.17 min and EIMS data at m/z 140 (M ), 125,
815.4210 [M+Na] , calcd for 815.4194). The compound displayed
42 carbon signals in its 13C NMR spectrum, of which 21 could be
ascribed to the aglycone moiety including two carbonyl carbons
at ı 210.9 and 172.4 (Table 1). A trisubstituted olefinic group at ı
139.1 and 121.3, two tertiary methyl carbons at ı 23.0 and 19.9, cou-
pled with the information from the 1H NMR spectrum [an olefinic
proton at ı 5.41, and two methyl proton singlet at ı 1.40 and 1.11]
(Table 2), indicated that the aglycone of 1 was similar to atrato-
genin A [8] except for the furan ring at C-13. The UV absorption at
206.5 nm in methanol suggested the presence of ␣,␤-unsaturanted
1
09 with the standard compound. The aqueous solution was neu-
+
tralized by an ion-exchange resin (DOWEX 500-8 H ), and then
filtered through filter paper to remove the resin. The filtrate was
concentrated under reduced pressure to dryness. The residue was
purified by silica gel CC with CHCl /MeOH (9:1) to give 12a.
3
2.7.1. Compound 12a
+
1
ESIMS (positive) m/z: 725 [M+Na] . H NMR (500 MHz, C5D5N):
.35 (1H, br.d, J = 5.3 Hz, H-6), 5.15 (1H, dd, J = 9.6, 2.3 Hz, cym I-H-1),
5

H. Bai et al. / Steroids 74 (2009) 198–207
205
Cynanoside P4 (4) was obtained as an amorphous powder. The
molecular formula was determined to be C48H74
O
by HRFABMS
19
+
1
13
(m/z 977.4755 [M+Na] , calcd for 977.4722). The H and C NMR
data suggested that 4 possessed the same aglycone as 3 and the
same sugar chain as 2. Enzymatic hydrolysis of 4 with naringinase
gave d-glucose and a deglucosyl derivative, which was identified
as 3. The ␤-d-glucopyranosyl moiety at cym II-C-4 was determined
by the HMBC correlation of ıH 4.94 (glc-H-1)/ıC 83.0 (cym II-C-
4
). Thus, the structure of 4 was established as (20R)-cynanogenin
C 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-
␣
-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Fig. 2. Key HMBC correlations of 1.
Cynanoside P5 (5) was obtained as an amorphous powder. Its
molecular formula, C49H76O20, was established by HRFABMS (m/z
+
1
007.4824 [M+Na] , calcd for 1007.4827). The NMR spectral com-
␥
-lactone moiety [17,18]. Observed HMBC correlations from ıH
parison of 5 and 2 revealed the superimposable signals, and the
difference was only observed in the signals due to the butenolide
moiety. Namely, the methine carbon at C-20 (ıC 80.6, ıH 5.42) in 2
was replaced by a downfield quaternary carbon at ı 110.1, and an
additional methoxyl moiety (ıC 50.6 and ıH 3.16) was observed.
The location of the methoxyl group at C-20 was deduced from
HMBC correlation of ıH 3.16 (H-22)/ıC 110.1 (C-20). The absolute
configuration of C-20 was determined to be 20S by the positive
cotton effect at 208.5 nm in the CD spectrum [17,22,23]. Thus,
the structure of 5 was established as (20S)-methoxycynanogenin
C 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-
␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
6
5
.17 (H-16) to ıC 172.4 (C-15), 176.4 (C-17) and 80.6 (C-20), ıH
.41 (H-20) to ıC 118.8 (C-16), 176.4 (C-17) and 20.1 (C-21), and
ıH 1.37 (H-21) to ıC 176.4 (C-17) and 80.6 (C-20), indicated that
the lactone moiety was ␥-methyl-substituted butenolide (Fig. 2).
The attachment of the butenolide moiety at C-13 was deduced
from the HMBC correlations of ıH 6.17 (H-16)/ıC 51.0 (C-13) and
ıH 1.40 (H-18)/ıC 176.4 (C-17). The NOESY correlations between
H-8 and H -18, H-8 and H -19, H -19 and H-2 indicated that all
3
3
3
of these protons and methyl groups were ␤ orientations. The ori-
entation of the 3-O-group was suggested to be ␤ by the coupling
constant between H-2 and H-3 (9.0 Hz). The absolute configuration
of C-20 was determined to be S-configuration by observation of a
Cynanoside Q1 (6) was obtained as an amorphous powder.
The molecular formula was determined to be C47H72O19 by
*
positive cotton effect at 202.5 nm (␲–␲ ) in the circular dichroic
+
spectroscopy [19,20,21]. Since the aglycone was a new structure,
it was named as (20S)-cynanogenin C. On acid hydrolysis, 1 gave
d-cymarose and l-diginose as component sugars [14]. The 1H NMR
spectrum suggested the presence of two ␤-cymaropyranosyl and a
HRFABMS (m/z 963.4571 [M+Na] , calcd for 963.4566). The com-
parison of 1H and C NMR data between 6 and cynanoside K
13
[10] suggested that they had the same sugar moiety but dif-
1
fered in the aglycone (Table 3). Further comparison of the
H
␣
-diginopyranosyl moieties with the anomeric proton resonances
at ı 5.21 (dd, J = 9.6, 1.8 Hz), 5.17 (br d, J = 2.4 Hz) and 5.13 (dd, J = 9.6,
.6 Hz), respectively. The sugar chain at C-3 of the aglycone was
and 13C NMR data assignable to the aglycone part revealed the
presence of one more hydroxyl group at C-13 of 6, which was
supported by the HMBC correlation from ıH 1.78, 1.74 (Ha-11
1
determined from the following HMBC correlations of ıH 5.13 (cym
II-H-1)/ıC 73.9 (dgn-C-4), ıH 5.17 (dgn-H-1)/ıC 82.1 (cym I-C-4),
and ıH 5.21 (cym I-H-1)/ıC 85.1 (C-3). Thus, the structure of 1 was
established as (20S)-cynanogenin C 3-O-␤-d-cymaropyranosyl-
and H -11) and 2.89, 1.97 (Ha-12 and H -12) to ı 76.3 (C-13).
b
b
C
Therefore, the aglycone of 6 could be 13-hydroxycynajapogenin
A [9]. Although the gross structure of 13-hydroxycynajapogenin
A has been established, the orientation of the hydroxyl group
at C-13 was ambiguous. The relative configuration of C-13 was
deduced from the 13C chemical shift of the neighboring car-
bons. It is known that ␥-carbon was very sensitive to steric
effect and conformational changes in molecules with hydroxyl
and other heteroatomic substitutions [24,25,26]. Since we have
recently isolated two glycosides, cynanoside K with cynajapogenin
A as the aglycone, and cynanoside M with 13-epi-cynajapogenin
A as the aglycone from C. atratum [10], a comparison of the
carbon chemical shift was carried out. C-8 and C-11 in 6 res-
onated 2.5 and 3.1 ppm upfield than those in cynanoside K,
whereas resonated 0.2 and 1.5 ppm downfield than those in
cynanoside M (Table 6). These indicated that C-8 and 13-OH
was ␥-gauch position, namely, the hydroxyl group of C-13 was
␤-orientation. The chemical shift changes were observed to be
small, which may be due to the ketone group at C-14 and
the distorted chair conformation of ring C [24,27]. Thus, the
structure of 6 was established as 13␤-hydroxycynajapogenin
A 3-O-␤-d-glycopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-
␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
(
1 → 4)-␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanoside P (2) was obtained as an amorphous powder.
2
The molecular formula was determined to be C48H74 O19 by
+
HRFABMS (m/z 977.4742 [M+Na] , calcd for 977.4722), which
was higher by 162 mass units than that of 1. Comparison
1
13
of the H and
C NMR data of 2 and 1 suggested that 2
had one more ␤-glucopyranosyl moiety than 1. Hydrolysis of
2
␤
with naringinase gave 1 and d-glucose. The position of the
-d-glucopyranosyl moiety in 2 was determined at cym II-C-4
from the glycosidation shift observed at cym II-C-3 (−0.7 ppm),
cym II-C-4 (+8.8 ppm) and cym II-C-5 (−1.5 ppm), and by the
HMBC correlation of ıH 4.95 (glc-H-1)/ıC 83.0 (cym II-C-4).
Thus, the structure of 2 was established as (20S)-cynanogenin
C 3-O-␤-d-glucopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-
␣
-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanoside P (3) was obtained as an amorphous powder. Its
3
molecular formula, C42H64O14 , was established by HRFABMS (m/z
+
8
15.4208 [M+Na] , calcd for 815.4193), which was the same as
1
13
that of 1. The H and C NMR of 3 was almost superimpos-
able with 1, except in the H NMR spectrum, H-20 of 3 was
1
Cynanoside Q2 (7) was obtained as an amorphous powder.
Its molecular formula C40H60O14 was determined from data of
shifted by + 0.14 ppm than that of 1, suggesting 3 would be a C-
+
2
0 epimer of 1. The CD spectrum of 3 showed a negative cotton
HRFABMS (m/z 787.3879 [M+Na] , calcd for 787.3881). The over-
effect at 205.0 nm, which was opposite to that of 1, indicating
R configuration of C-20. Thus, the structure of 3 was established
as (20R)-cynanogenin C 3-O-␤-d-cymaropyranosyl-(1 → 4)-␣-l-
diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
all structure assignment was accomplished by a combination of 1D
and 2D NMR spectra. The 1H and C NMR spectra indicated that
7 possessed the same aglycone as 6, and the same sugar moiety
as cynanoside H [14]. Thus, the structure of 7 was established as
13

2
06
H. Bai et al. / Steroids 74 (2009) 198–207
Table 6
Part 13 C NMR data (125 HMz) of 6, cynanoside K and cynanoside M in C5D5N.
C atom
6
Cynanoside K
Cynanoside M
ꢁ = ı6 − ıcynanoside K
ꢁ = ı6 − ıcynanoside M
8
9
43.0
52.4
22.4
39.8
76.3
45.5
52.0
25.5
33.8
47.9
209.4
42.8
50.5
20.9
30.0
46.4
212.0
−2.5
0.2
1.9
1.5
9.8
29.9
1.1
0.4
1
1
−3.1
6.0
28.4
3.7
1
2
1
3
14
213.1
1
␤
3␤-hydroxycynajapogenin A 3-O-␣-l-oleandropyranosyl-(1 → 4)-
B 3-O-␤-d-glycopyranosyl-(1 → 4)-␤-d-cymaropyranosyl-(1 → 4)-
␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
-d-digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanoside Q (8) was obtained as an amorphous powder. The
Cynanoside S (12) was obtained as an amorphous powder. Its
3
molecular formula was determined to be C46H70O19 by HRFABMS
molecular formula was determined to be C40H60
O
16
13
by HRFABMS
+
1
13
+
(
m/z 949.4438 [M+Na] , calcd for 949.4409). From its H and
C
(m/z 819.3787 [M+Na] , calcd for 819.3779). The C NMR spec-
trum (Table 5) in combination with analysis of the DEPT and HMQC
spectra revealed 40 carbon signals due to six quaternary carbons,
19 methines, seven methylenes and eight methyls, of which 19 were
assigned to the aglycone part including two carbonyl carbons at ı
175.7 and 164.0, and the remaining 21 were ascribed to the sugar
moiety. The 1H NMR spectrum of the aglycone moiety showed two
aromatic protons at ı 7.24 (d, J = 2.1 Hz, H-15) and 6.99 (d, J = 2.1 Hz,
H-16), suggesting the presence of a 2.3-disubstituents furan ring.
Also, in the 1H NMR spectrum, signals assignable to carbinylic pro-
tons of the aglycone were observed at ı 5.43 (ddd, J = 11.4, 9.6 and
5.7 Hz), 4.04 (ddd, J = 11.7, 8.8 and 4.6 Hz) and 3.62 (ddd, J = 11.5, 8.8
and 5.5 Hz), indicating those carbinylic protons could all be placed
in the axial-orientations. The partial structures of the aglycone (C-
1 to C-4, C-6 to C-11 and C-15 to C-16) were deduced from the
detailed analyses of the DQFCOSY spectrum (Fig. 3). Interpretation
of the HMBC spectral data led to the connectivity of those three par-
tial units and quaternary carbons to construct the structure of the
aglycone. The HMBC correlations from ıH 2.50 (H-4) to ıC 119.5 (C-
6), ıH 5.27 (H-6) to ı 40.2 (C-10), as well as from ıH 1.04 (H-19) to ı
NMR data, it was apparent that 8 possessed one more glucopyra-
nosyl moiety with the anomeric proton signal at ı 5.27 (d, J = 7.8 Hz)
than 7. Enzymatic hydrolysis of 8 gave 7 and d-glucose. The attach-
ment of the glucopyranosyl moiety to ole-C-4 was determined by
the HMBC correlation of ıH 5.27 (glc-H-1)/ıC 82.2 (ole-C-4). Thus,
the structure of 8 was established as 13␤-hydroxycynajapogenin A
3
␤
-O-␤-d-glycopyranosyl-(1 → 4)-␣-l-oleandropyranosyl-(1 → 4)-
-d-digitoxopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanoside R (9) was obtained as an amorphous powder. The
1
molecular formula was determined to be C42
H
O13 by HRFABMS
62
1
+
(
m/z 797.4117 [M+Na] , calcd for 797.4088). The H NMR spectrum
showed a set of signals due to a furan ring with 2,3-disubstituents
at ı 7.46 (d, J = 1.8 Hz) and 6.42 (d, J = 1.8 Hz), a trisubstituted
olefinic proton at ı 5.56 (br s), a carbinylic proton at ı 4.79 (dd,
J = 12.5, 7.3 Hz), and three methyl proton singlets at ı 2.24, 1.49
and 1.08 (Table 4). A comparison of the 13C NMR spectrum of 9
and 6, suggested that their resonances were different in A and
C rings. Namely, a carbinylic carbon (ı 70.0, C-2) in 6 was sub-
stituted by a carbonyl carbon (ı 205.6), and a hydroxyl group
located at C-13 of 6 was substituted by a methyl group (ı 23.9).
These NMR characteristics indicated that the aglycone of 9 should
be atratogenin B [8]. On acid hydrolysis, 9 afforded d-cymarose
and l-diginose as component sugars. The ␤-d-cymaropyranosyl-
C
C
45.2 (C-1), 140.2 (C-5) and 47.5 (C-9) indicated the connectivity of
the A ring and B ring (Fig. 3). Further evidence of the di-substituted
furan ring was also provided by the HMBC correlations from ıH
7.24 (H-15) to ıC 114.3 (C-17) and 159.6 (C-20), ıH 6.99 (H-16) to ıC
(
1 → 4)-␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoyl moiety
159.6 (C-20), and ıH 2.57 (H-21) to ı 114.3 (C-17) and 159.6 (C-20).
A carboxylic acid unit was attached at C-11 on the basis of the cor-
C
connected to C-3 was established by detailed analysis of 2D NMR
spectra and comparison of the sugar data with 1. Thus, the structure
of 9 was established as atratogenin B 3-O-␤-d-cymaropyranosyl-
relation between ıH 2.78, 2.70 (H-11) and ı 175.7 (C-12). Although
C
the correlations from ıH 5.43 (H-8) and 6.99 (H-16) to ı 164.0
C
(
1 → 4)-␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
(C-13) though 3JCH was not observed, the ester carbon at ı 164.0
was assigned to C-13 on the basis of the molecular formula and the
degree of unsaturation. Alkaline hydrolysis of 12 with 0.28% NaOMe
in MeOH gave 12a and 12b (methyl 2-methylfuran-3-carboxylate)
(Scheme 1), indicating that the furan ring and C-8 were connected
through an ester bond. Careful comparison of the chemical shift
of C-20 of 12 and cyanosides A-C [14] also supported the above
result. The relative stereochemistry of the aglycone was elucidated
by the NOESY correlations. Observed 1,3-diaxial NOE correlations
Cynanoside R (10) was obtained as an amorphous pow-
2
der. Its molecular formula C42H62O14 was determined from data
of HRFABMS (m/z 813.4062 [M+Na] , calcd for 813.4037). The
+
NMR spectral comparison of 10 and 9 suggested the super-
imposable signals, and the difference was only observed in
the ring A of the aglycone moiety. Namely, the methylene
assignable to C-1 of 9 (ı 52.0) was replaced by an oxygenated
methine (ı 83.5) in 10. The ␣-orientation of H-1 was deter-
mined by observed 1,3-diaxial NOE correlations for H-1/H-3
and H-1/H-9. Thus, the structure of 10 was established as
for H-2/H -19, H-1␣/H-3 revealed the axial-orientation of H-2, H-3
3
and H -19. The ␤ orientation of H-8 and ␣ orientation of H-9 were
3
1
␤-hydroxyatratogenin B 3-O-␤-d-cymaropyranosyl-(1 → 4)-␣-l-
diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanoside R (11) was obtained as an amorphous powder.
3
The HRFABMS of 11 suggested the molecular formula of C48H72O19
+
(
1
m/z 975.4541 [M+Na] , calcd for 975.4565), which was higher by
62 mass units than that of 10. Comparing the 1H and C NMR
13
data of 11 with those of 10, 11 had one more ␤-glucopyranosyl
moiety with the anomeric proton signal resonating at ı 4.93 (d,
J = 7.8 Hz). Enzymatic hydrolysis of 11 gave 10 and d-glucose. The
position of the glucopyranosyl moiety at cym II-C-4 was suggested
by the HMBC correlation of ıH 4.93 (glc-H-1)/ıC 83.0 (cym II-C-4).
Thus, the structure of 11 was established as 1␤-hydroxyatratogenin
Fig. 3. Key DQFCOSY and HMBC correlations of 12.

H. Bai et al. / Steroids 74 (2009) 198–207
207
Scheme 1. Alkaline hydrolysis of 12.
suggested by the NOESY correlations for H-8/H -19, H-1␣/H-9.
[9] Lee KY, Sung HS, Kim YC. New acetylcholinesterase-inhibitory pregnane glyco-
sides of Cynanchum atratum roots. Helv Chim Acta 2003;86:474–83.
10] Bai H, Li W, Koike K. Pregnane glycosides from Cynanchum atratum. Steroids
3
Therefore, the aglycone of 12 was determined to be a new struc-
ture and named cynanogenin D. The type of the sugar units and
the sequence of the oligosaccharide chain of 12 were established
by a combination of 2D NMR experiments and the result of the acid
hydrolysis, which was the same as that of 1. Thus, the structure of
[
2008;73:96–103.
[11] Zhang ZX, Zhou J, Hayashi H, Mitsuhashi H. Study on the constituents of Ascle-
piadaceae Plants. LVIII. The structures of five glycosides, Cynatratoside-A, -B, -C,
-
D, and -E, from the Chinese drug “Pai-Wei,” Cynanchum atratum Bunge. Chem
Pharm Bull 1985;33:1507–14.
12 was established as cynanogenin D 3-O-␤-d-cymaropyranosyl-
[12] Zhang ZX, Zhou J, Hayashi K, Mitsuhashi H. Study on the constituents of
Asclepiadaceae Plants. LXI. The structure of Cynatratoside-F from the Chinese
drug “Pai-Wei,” dried root of Cynanchum atratum Bunge. Chem Pharm Bull
(
1 → 4)-␣-l-diginopyranosyl-(1 → 4)-␤-d-cymaropyranoside.
Cynanosides P –P5 (1–5), Q –Q (6–8), R –R (9–11) and
1
1
3
1
3
1985;33:4188–92.
cynanoside S (12) were evaluated for their cytotoxic activities
against human HL-60 myeloid leukemia, human stomach KATO-
III adenocarcinoma and human lung A549 adenocarcinoma cells.
Compounds 9 and 10 showed cytotoxic activities against HL-60 cells
with IC50 values of 29.47 ± 5.38 and 52.26 ± 13.38 ␮M, as compared
to the activity of etoposide, which was used as a positive control
[13] Day SH, Wang JP, Won SJ, et al. Bioactive constituents of the roots of Cynanchum
atratum. J Nat Prod 2001;64:608–11.
[
14] Bai H, Li W, Koike K, et al. Cynanosides A-J, ten novel pregnane glycosides from
Cynanchum atratum. Tetrahedron 2005;61:5797–811.
[
15] Warashina T, Noro T. Glycosides of 14,15-seco and 13,14:14,15-disecopregnanes
from the roots of Cynanchum sublanceolatum. Chem Pharm Bull
2
006;54:1551–60.
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16] Tai Y, Cao X, Li X, Pan Y. Identification of C-21 steroidal glycosides from the
roots of Cynanchum chekiangense by high-performance liquid chromatogra-
phy/tandem mass spectrometry. Anal Chim Acta 2006;572:230–6.
(
IC50 0.29 ± 0.02 ␮M). All compounds were inactive for KATO-III
adenocarcinoma and A549 adenocarcinoma cells at 100 ␮M.
[
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and absolute configuration of heterosubtituted 2(5H)-furanones. Chirality
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This study was partially supported by the National Natural Sci-
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