Lanostane-type triterpenoids from the roots of Kadsura coccinea

Article abstract of DOI:10.1021/np7007522

Seven new lanostane-type triterpenoids, seco-coccinic acids A - F (1-6) and coccinilactone A (7), were isolated from the roots of Kadsura coccinea. Their structures were established on the basis of spectroscopic data analysis. The absolute configuration at C-24 of compound 5 was confirmed by the modified Mosher's method. The cell growth inhibitory effects of these compounds were determined in human leukemia HL-60 cells, and it was found that compounds 1, 2, 3, and 5 exhibited antiproliferative effects with GI₅₀ values ranging from 6.8 to 42.1 7μM.

Full text of DOI:10.1021/np7007522

990
J. Nat. Prod. 2008, 71, 990–994
Lanostane-Type Triterpenoids from the Roots of Kadsura coccinea
Nan Wang,† Zhanlin Li,† Dandan Song,‡ Wei Li,§ Hongwei Fu,§ Kazuo Koike,§ Yuehu Pei,† Yongkui Jing, and
Huiming Hua*,†
Shenyang Pharmaceutical UniVersity, Shenyang 110016, People’s Republic of China, Toho UniVersity, Funabashi, Chiba 274-8510, Japan, and
Mount Sinai School of Medicine, New York, New York 10029
ReceiVed December 29, 2007
Seven new lanostane-type triterpenoids, seco-coccinic acids A-F (1-6) and coccinilactone A (7), were isolated from
the roots of Kadsura coccinea. Their structures were established on the basis of spectroscopic data analysis. The absolute
configuration at C-24 of compound 5 was confirmed by the modified Mosher’s method. The cell growth inhibitory
effects of these compounds were determined in human leukemia HL-60 cells, and it was found that compounds 1, 2, 3,
and 5 exhibited antiproliferative effects with GI50 values ranging from 6.8 to 42.1 µM.
Lanostane-type triterpenoids have been isolated from members
of the genus Kadsura, such as K. japonica,1 K. ananosma,2,3 K.
coccinea,4 K. heteroclita,5–8 K. longipedunculata,9–12 and K.
lancilimba.13 Several of these triterpenoids have been found to have
potential anti-HIV, anticancer, and cholesterol biosynthesis inhibi-
tory activities. For example, schisanlactone E and changnanic acid
isolated from K. longipedunculata have been found to have an
antiproliferative effect against murine leukemia P388 cells,12 and
ananosic acids B and C isolated from K. ananosma exhibited
cytotoxicity against human CCRF-CEM cells and HeLa cells.2
Kadsura coccinea (Lem.) A. C. Sm. (Schizandraceae) is dis-
tributed widely in the southern part of mainland China. The dried
roots of K. coccinea, called “Heilaohu” in Chinese,14 are used as
a folk medicine for the treatment of rheumatoid arthritis and for
gastric and duodenal ulcers.15 The isolation and structure elucidation
of seven new lanostane-type triterpenoids (1-7) from the air-dried
roots of K. coccinea are described herein, and the antiproliferative
effects of these lanostane-type triterpenoids were determined against
human leukemia HL-60 cells. Compounds 1, 2, 3, and 5 as well as
the crude chloroform extract showed antiproliferative effects.
carboxylic group at δ 176.9, one terminal double bond at δ 149.7
and 112.2, and a ketone carbonyl group at δ 210.2. On the basis of
the above data, compound 1 was determined as a lanostane-type
triterpenoid.17 The H and 13C NMR data were assigned from the
1
1H-1H COSY, HMQC, and HMBC spectra (Tables 1 and 2). The
presence of a seco-ring A was demonstrated by signals at δ 176.9
(C-3), 149.7 (C-4), and 112.2 (C-28).18 The occurrence of a terminal
double bond in 1 was shown by the two broad singlets at δ 4.99
1
and 4.98 in the H NMR spectrum. In the HMBC spectrum, the
correlations between C-5 (δ 45.7) and H-28 (δ 4.99 and 4.98), H-19
(δ 0.93), and H-29 (δ 1.85) implied that the terminal double bond
was located between C-4 and C-28. A trisubstituted olefinic proton
at δ 5.32 showed long-range correlations with C-5 (δ 45.7) and
C-6 (δ 29.7), indicating that a double bond is located between C-7
and C-8. The carbonyl group signal at δ 210.2 had correlations
with H-22 (δ 2.47 and 2.18) and H-24 (δ 2.31 and 2.29), indicating
it to occur at C-23, which was further supported by a prominent
peak at m/z 57 (C4H9) and 85 (C5H9O) in the EIMS. In a NOESY
experiment, H-9 at δ 2.64 showed correlations with CH3-18 at δ
0.82 and CH3-19 at δ 0.93, which indicated that H-9 is ꢀ-oriented.
On the basis of the above spectroscopic data, compound 1 (seco-
coccinic acid A) was established as 23-oxo-3,4-seco-9ꢀH-lanost-
4(28),7-dien-3-oic acid.
Results and Discussion
The ethanol extract of the air-dried roots of K. coccinea was
partitioned with petroleum ether, CHCl3, EtOAc, and n-BuOH,
successively. Six new lanostane-type triterpenoids (1-6) were
obtained from the petroleum ether extract through a series of
chromatographic separations, while a new triterpenoid (7) was
isolated from the CHCl3 extract of K. coccinea. The structures of
1-7 were elucidated on the basis of spectroscopic methods.
Compound 1 was obtained as colorless needles, and its molecular
formula was determined as C30H48O3 on the basis of HRFABMS
(m/z 457.3674 [M + H]+). The FABMS showed a characteristic
fragmentation for a 3,4-seco-triterpenoid with a prominent peak at
m/z 383 [M - CH2CH2COOH]+.16 The IR spectrum of compound
1 indicated the presence of a carbonyl (1710 cm-1) group. The 1H
NMR spectrum showed four methyl singlets and three methyl
doublets at δ 0.97 (J ) 5.8 Hz), 0.92 (J ) 6.7 Hz), and 0.91 (J )
6.4 Hz). The 13C NMR spectrum exhibited 30 carbon signals that
were sorted by a DEPT experiment as seven methyls, 10 methyl-
enes, six methines, and seven quaternary carbons, including one
Compounds 2-4 were all obtained as colorless needles. The 1H
NMR spectra of these three substances showed six methyl singlets
and a methyl doublet, and the 13C NMR spectra revealed 30 carbon
signals in all cases. Except for their side chains, the proton and
carbon NMR data of compounds 2-4 were similar to those of
compound 1, including resonances at δ 176.9 (C-3), 149.7 (C-4),
and 112.2 (C-28) characteristic of a seco-ring A unit.18 On the basis
of these spectroscopic data, compounds 2-4 were found to be
identical with 1 in their ring A-D portions.
The molecular formula of compound 2 was determined as
C30H46O3 on the basis of its HRFABMS (m/z 455.3539 [M + H]+).
The IR spectrum showed the presence of a conjugated carbonyl
(1680 cm-1) and a carboxylic acid (1711 cm-1) group. Compound
2 exhibited differences from compound 1 in the NMR spectra with
the appearance of two olefinic carbon signals at δ 123.8 and 153.9
in the 13C NMR spectrum and two methyl singlets at δ 2.22 and
1.77 in the 1H NMR spectrum, confirming the presence of a double
bond at C-24. In the HMBC spectrum of compound 2, a conjugated
carbonyl group signal at δ 201.8 had correlations with H-22 (δ
2.56 and 2.18) and H-24 (δ 6.18), indicating a ketone carbonyl
group at C-23. On the basis of these data, the structure of compound
2 was assessed as a triterpenoid with a 23-keto-∆24 side chain. Thus,
2 (seco-coccinic acid B) was assigned as 23-oxo-3,4-seco-9ꢀH-
lanost-4(28),7,24-trien-3-oic acid.
* Corresponding author. Tel: +862423986465. Fax: +862423986465.
E-mail: huimhua@163.com.
† School of Traditional Chinese Materia Medica, Shenyang Pharmaceuti-
cal University.
‡ School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical
University.
§ Toho University.
Mount Sinai School of Medicine.
10.1021/np7007522 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/21/2008

Lanostane-Type Triterpenoids from Kadsura coccinea
Journal of Natural Products, 2008, Vol. 71, No. 6 991
Table 1. 1H NMR Data (500 MHz) of Compounds 1-7 (δH, pyridine-d5, J in Hz)
H
1
2
3
4
5
6
7
1
2.03 (1H, m)
1.99 (1H, m)
2.57 (2H, m)
2.03 (1H, m)
1.98 (1H, m)
2.57 (2H, m)
2.02 (1H, m)
1.97 (1H, m)
2.56 (2H, m)
2.03 (1H, m)
1.92 (1H, m)
2.57 (2H, m)
2.03 (1H, m)
1.96 (1H, m)
2.57 (2H, m)
2.76 (2H, m)
1.90 (2H, m)
2
5
6
2.37 (1H, m)
2.11 (1H, m)
2.19 (1H, m)
2.78 (2H, m)
1.66 (1H, m)
1.54 (2H, m)
2.26
(1H, d, 7.1)
2.38 (1H, m)
2.26
2.25
2.26
(1H, d, 6.9)
2.40 (1H, m)
2.26
(1H, d, 7.1)
2.40 (1H, m)
(1H, d, 7.1)
2.39 (1H, m)
2.09 (1H, m)
(1H, d, 7.1)
2.38 (1H, m)
2.06 (1H, m)
1.92 (1H, m)
2.07 (1H, m)
5.32
(1H, d, 2.5)
2.07 (1H, m)
5.32
(1H, d, 3.5)
2.08 (1H, m)
5.31
(1H, d, 2.8)
1.73 (1H, m)
1.50 (2H, m)
7
5.31
(1H, d, 2.8)
5.3
(1H, d, 3.0)
1.87 (2H, m)
2.14 (1H, m)
5.26 (1H, d, 5.8)
2.09 (1H, m)
8
9
11
2.12 (1H, m)
2.64 (1H, m)
1.69 (1H, m)
1.54 (1H, m)
1.81 (1H, m)
2.64 (1H, m)
1.68 (1H, m)
1.55 (1H, m)
1.81 (1H, m)
2.63 (1H, m)
1.68 (1H, m)
1.52 (1H, m)
1.80 (1H, m)
2.65 (1H, m)
1.53 (2H, m)
2.64 (1H, m)
1.67 (1H, m)
1.55 (1H, m)
1.84 (1H, m)
5.58
(1H, d, 5.5)
2.08 (1H, m)
12
1.77 (1H, m)
1.62 (1H, m)
1.64 (1H, m)
1.56 (1H, m)
1.44 (1H, m)
1.90 (1H, m)
1.24 (1H, m)
1.53 (1H, m)
0.82 (3H, s)
0.93 (3H, s)
2.17 (1H, m)
0.97
1.64 (1H, m)
1.54 (1H, m)
1.46 (1H, m)
1.93 (1H, m)
1.29 (1H, m)
1.55 (1H, m)
0.82 (3H, s)
0.93 (3H, s)
2.18 (1H, m)
1.01
1.64 (1H, m)
1.54 (1H, m)
1.45 (1H, m)
1.90 (1H, m)
1.26 (1H, m)
1.53 (1H, m)
0.81 (3H, s)
0.93 (3H, s)
2.19 (1H, m)
1.01
1.64 (1H, m)
1.55 (1H, m)
1.44 (1H, m)
1.97 (1H, m)
1.30 (1H, m)
1.52 (1H, m)
0.82 (3H, s)
0.94 (3H, s)
1.50 (1H, m)
0.96
1.87 (1H, m)
1.36 (2H, m)
1.89 (1H, m)
1.36 (2H, m)
15
16
1.54 (1H, m)
1.46 (1H, m)
1.96 (2H, m)
1.28 (1H, m)
1.53 (1H, m)
0.81 (3H, s)
0.94 (3H, s)
1.52 (1H, m)
0.95
1.52 (2H, m)
1.55 (2H, m)
17
18
19
20
21
1.63 (1H, m)
0.70 (3H, s)
1.11 (3H, s)
1.47 (1H, m)
0.97
1.66 (1H, m)
0.72 (3H, s)
1.16 (3H, s)
2.20 (1H, m)
1.01
(3H, d, 5.8)
2.47 (1H, m)
(3H, d, 5.7)
2.56 (1H, m)
(3H, d, 6.4)
2.70 (1H, m)
2.40 (1H, m)
(3H, d, 5.8)
2.28 (1H, m)
(3H, d, 5.8)
1.40 (1H, m)
(3H, d, 6.4)
1.60 (1H, m)
(3H, d, 5.8)
2.18 (1H, m)
22
2.18 (1H, m)
2.18 (1H, m)
2.25 (1H, m)
5.92 (1H, m)
0.97 (1H, m)
1.93 (1H, m)
1.69 (1H, m)
4.37 (1H, m)
1.20 (1H, m)
2.34 (1H, m)
2.17 (1H, m)
7.21 (1H, m)
2.48 (1H, m)
23
24
2.31 (1H, m)
2.29 (1H, m)
6.18 (1H, s)
2.8
5.93 (1H, m)
2.31 (2H, m)
(2H, d, 7.1)
1.51 (3H, s)
1.51 (3H, s)
25
26
2.24 (1H, m)
0.92
(3H, d, 6.7)
0.91
2.24 (1H, m)
0.94
(3H, d, 6.4)
0.92
1.77 (3H, s)
2.22 (3H, s)
1.55 (3H, s)
1.55 (3H, s)
5.29 (1H, s)
4.98 (1H, s)
1.92 (3H, s)
27
2.13 (3H, s)
(3H, d, 6.4)
4.99 (2H, s)
1.85 (3H, s)
1.07 (3H, s)
(3H, d, 6.7)
1.39 (3H, s)
1.42 (3H, s)
0.78 (3H, s)
28
29
30
4.99 (2H, s)
1.84 (3H, s)
1.06 (3H, s)
4.97 (2H, s)
1.84 (3H, s)
1.04 (3H, s)
5.00 (2H, s)
1.86 (3H, s)
1.04 (3H, s)
5.00 (2H, s)
1.86 (3H, s)
1.05 (3H, s)
4.97 (2H, s)
1.83 (3H, s)
0.80 (3H, s)
Compound 3 gave a [M + Na]+ ion in the HRFABMS at m/z
495.3438, consistent with a molecular formula of C30H48O4. It
showed IR absorptions for hydroxyl, ketone carbonyl, and car-
boxylic acid groups at 3457, 1723, and 1700 cm-1, respectively.
Compound 3 exhibited an oxygenated carbon signal at δ 69.5 in
the 13C NMR spectrum, confirming the presence of a hydroxyl
group in the side chain. In the HMBC spectrum, the signal appearing
at δ 69.5 had long-range correlations with H-24 (δ 2.80) and the
methyl protons of H-26 (δ 1.51) and H-27 (δ 1.51), which suggested
a hydroxyl group was located at C-25. The carbonyl carbon signal
resonating at δ 211.3 was assigned to C-23 due to the long-range
correlation with the methylene protons of H-22 (δ 2.28 and 2.25)
and H-24 (δ 2.80). Thus, 3 (seco-coccinic acid C) was established
as 25-hydroxy-23-oxo-3,4-seco-9ꢀH-lanost-4(28),7-dien-3-oic acid.
Compound 4 showed a quasimolecular ion peak [M + H]+ at
m/z 457.3639 (C30H48O3) in the HRFABMS. The IR spectrum
showed the presence of a hydroxyl (3428 cm-1) and a carboxylic
acid (1705 cm-1) group. Its 13C NMR spectrum displayed an
oxygenated carbon at δ 69.7 and olefinic carbons at δ 124.6 and
141.7. The carbon signal at δ 69.7 was assigned to C-25 due to
long-range correlations with the methyl protons of H-26 (δ 1.55)
and H-27 (δ 1.55) in the HMBC spectrum. The HMBC correlations
between the signal at δ 141.7 and H-22 (δ 2.28), H-26 (δ 1.55),
and H-27 (δ 1.55) and between the signal at δ 124.6 and H-22 (δ
2.28) indicated that a double bond is located at C-23. Accordingly,
4 (seco-coccinic acid D) was elucidated as 25-hydroxy-3,4-seco-
9ꢀH-lanost-4(28),7,23-trien-3-oic acid.
Compound 5 was obtained as colorless needles with a molecular
formula of C30H48O3, established on the basis of the HRFABMS
(m/z 479.3495 [M + Na]+). The 1H NMR spectrum of compound
5 showed signals of five methyl singlets and a methyl doublet at δ
0.96 (J ) 5.8 Hz, CH3-21). The 13C NMR spectrum revealed 30
carbon signals, which were sorted by a DEPT experiment as six
methyls, 11 methylenes, six methines, and seven quaternary carbons,
including one oxygenated methine, one carboxylic acid group, and
two terminal double bonds. Compound 5 was found to be identical
with compound 1 in the 1H and 13C NMR data except for the side
chain. In the HMBC spectrum, the long-range correlations between
the oxygenated carbon signal at δ 75.7 and H-23 (δ 1.93 and 1.69),
H-26 (δ 5.29 and 4.98), and H-27 (δ 1.92) suggested a hydroxyl
group located at C-24. The olefinic carbon at δ 110.1 showed
HMBC correlations with proton signals at δ 4.37 (H-24) and 1.92
(H-27), indicating that another terminal double bond was located
at C-26. The NOESY correlations of H-9/H-18, H-9/H-19, and H-9/
H-29 indicated that H-9 is ꢀ-oriented. The configuration of the chiral
center C-24 was determined through the NMR study of MTPA
esters of the methyl ester 5a. In the 1H NMR spectrum of the (R)-
MTPA ester 5b, H2-26 and H3-27 appeared deshielded, whereas
H2-23 and H1-20 were shielded, in comparison to the analogous
data for (S)-MTPA ester 5c (Figure 2). Thus, the configuration at

992 Journal of Natural Products, 2008, Vol. 71, No. 6
Wang et al.
Table 2. 13C NMR Data (125 MHz) of Compounds 1-7 (δC,
pyridine-d5)
Table 3. GI50 Values of Triterpenoids That Inhibit HL-60 Cell
Growtha
C
1
2
3
4
5
6
7
compound
GI50 ( SE (µM)
1
2
3
4
5
6
7
8
30.0 t
29.9 t
30.0 t
30.0 t
30.0 t
30.0 t
30.0 t
30.0 t
30.0 t
30.0 t
29.9 t
33.4 t
34.9 t
32.3 t
1
2
3
5
6.8 ( 1.36
13.3 ( 0.99
12.1 ( 2.37
42.1 ( 10.2
3.8 ( 0.48
176.9 s 176.9 s 177.0 s 176.9 s 176.9 s 176.8 s 174.4 s
149.7 s 149.7 s 149.5 s 149.5 s 150.4 s 148.2 s
45.7 d
29.7
118.3 d 118.3 d 118.3 d 118.2 d 118.2 d
146.9 s 147.0 s 146.9 s 147.1 s 147.1 s
85.9 s
52.9 d
25.7 t
28.4 t
42.2 d
45.7 d
29.7 t
45.7 d
29.7 t
45.7 d
29.7 t
45.7 d
29.7 t
49.4 d
28.2 t
27.0 t
42.7 d
5-FU
t
a GI50 is the concentration that inhibited 50% of cell growth. The
data shown are means ( SE of three independent experiments.
9
39.2 d
36.7 s
18.9 t
34.1 t
44.0 s
51.9 s
34.4 t
28.6 t
53.2 d
21.8 q
24.3 q
33.1 d
19.7 q
50.4 t
39.2 d
36.7 s
18.9 t
34.1 t
44.0 s
51.9 s
34.4 t
28.6 t
53.5 d
21.8 q
24.3 q
33.7 d
19.7 q
51.7 t
39.1 d
36.7 s
18.9 t
34.1 t
44.0 s
51.9 s
34.4 t
28.6 t
53.2 d
21.8 q
24.3 q
33.0 d
19.8 q
52.1 t
39.2 d
36.7 s
18.9 t
34.1 t
43.9 s
51.8 s
34.5 t
28.5 t
53.0 d
21.9 q
24.3 q
36.9 d
18.6 q
39.3 t
39.2 d 143.0 s 147.2 s
36.7 s 42.9 s 42.2 s
18.9 t 118.8 d 117.4 d
C-10 (δ 42.9) and C-13 (δ 44.2), indicating that a double bond is
located between C-9 and C-11. Proton signals of H-23 (δ 2.34 and
2.17) and H-27 (δ 2.13) were correlated with an olefinic carbon at
δ 142.5, which implied that another double bond was located
between C-24 and C-25. A carboxylic acid group was determined
at C-26 by the HMBC correlations between the signal at δ 170.7
and an olefinic proton at δ 7.21 (H-24) and a methyl proton at δ
2.13 (H-27). The relative configuration of compound 6 was
determined by the NOESY spectrum, in which H-27 showed
correlation with H-23 but no NOE correlation was observed between
H-27 and H-24, indicating that the double-bond geometry was in
the E configuration. The H-8 signal showed NOE correlations with
H-18, H-19, and H-29, respectively. Accordingly, compound 6
(seco-coccinic acid F) was assigned as 24(E)-3,4-seco-8ꢀH-lanost-
4(28),9(11),24-triene-3,26-dioic acid.
Compound 7 was obtained as colorless needles, and its molecular
formula of C30H48O3 was established by the HRFABMS (m/z
457.3685 [M + H]+). The 1H NMR spectrum showed five methyl
singlets and three methyl doublets at δ 1.01 (J ) 5.8 Hz, CH3-21),
0.94 (J ) 6.4 Hz, CH3-26), and 0.92 (J ) 6.7 Hz, CH3-27).
Altogether 30 carbon signals were revealed in the 13C NMR
spectrum, which were sorted by a DEPT experiment as eight
methyls, nine methylenes, six methines, and seven quaternary
carbons, including one oxygenated quaternary carbon at δ 85.9 and
a carbonyl carbon at δ 174.4, suggesting compound 7 possesses a
lactone ring unit.17 Compound 7 was found to be identical with
compound 1 in the side chain, including a ketone located at C-23.
In the HMBC spectrum, H-28 (δ 1.39), H-29 (δ 1.42), and H-5 (δ
1.66) were correlated with C-4 (δ 85.9), and a ketone carbon at
C-3 (δ 174.4) showed correlations with the H-1 (δ 1.90) and H-2
(δ 2.78) methylene protons. A trisubstituted olefinic proton at δ
5.26 showed long-range correlations with C-12 (δ 37.6) and C-10
(δ 42.2), indicating that a double bond is located between C-9 and
C-11. The stereochemistry of compound 7 was determined by its
NOESY spectrum, in which correlations of H-8/H-18, H-8/H-19,
and H-8/H-28 suggested that H-8 is ꢀ-oriented. Thus, the structure
of 7 was assigned as 23-oxo-8ꢀH-lanost-9(11)-en-3,4-olide, and
this compound was given the trivial name coccinilactone A.
The growth inhibitory effects of these compounds against HL-
60 human leukemia cells were determined. Compounds 1, 2, 3,
and 5 showed inhibitory activity, while compound 1 was the most
effective, with a GI50 value of 6.8 µM. The GI50 values of these
four compounds are shown in Table 3. The crude chloroform extract
showed inhibitory effect on HL-60 cell growth with a GI50 value
of 16.1 ( 4.89 µg/mL.
10
11
12
13
14
15
16
17
18
19
20
21
22
34.3 t
44.0 s
51.9 s
34.4 t
28.6 t
53.5 d
21.9 q
24.3 q
36.4 d
18.8 q
32.5 t
32.8 t
37.9 t
44.2 s
47.5 s
33.9 t
28.2 t
51.2 d
14.9 q
27.2 q
36.3 d
18.4 q
35.5 t
37.6 t
44.3 s
47.5 s
34.0 t
27.4 t
51.5 d
14.7 q
23.7 q
33.0 d
19.7 q
50.7 t
23 210.2 s 200.7 s 211.3 s 124.6 d
26.0 t 210.2 s
24
25
26
27
52.5 t
24.7 d 153.9 s
22.7 q
22.6 q
123.8 d
56.0 t 141.7 d
75.7 d 142.5 d
52.5 t
24.7 t
22.6 q
22.7 q
25.4 q
33.0 q
18.6 q
69.5 s
30.4 q
30.2 q
69.7 s 149.5 s 129.0 s
30.9 q 110.1 t 170.7 s
30.9 q
27.3 q
20.6 q
18.2 q
12.9 q
28 112.2 t
29
30
112.2 t 112.1 t 112.2 t 112.2 t 114.1 t
26.0 q
27.6 q
26.0 q
27.5 q
26.0 q
27.5 q
26.0 q
27.5 q
26.0 q
27.6 q
23.6 q
18.5 q
C-24 was concluded as R.19 Thus, 5 (seco-coccinic acid E) was
assigned as 24R-hydroxy-3,4-seco-9ꢀH-lanost-4(28),7,25(26)-trien-
3-oic acid.
Compound 6 was obtained as colorless needles, and its molecular
formula was determined as C30H46O4 on the basis of its HRFABMS
(m/z 471.3472 [M + H]+). The H NMR spectrum showed five
1
methyl singlets and a methyl doublet at δ 0.97 (J ) 6.4 Hz, CH3-
21). The 13C NMR spectrum revealed 30 carbon signals, which
were sorted by the DEPT experiment as six methyls, 10 methylenes,
six methines, and eight quaternary carbons. The signals in the 13
C
NMR spectrum at δ 176.8 (C-3) and 170.7 (C-26) and the
absorbance band at 1698 cm-1 in the IR spectrum showed the
1
presence of two carboxylic acid groups. The H NMR spectrum
revealed two trisubstituted olefinic protons at δ 5.58 (d, J ) 5.5
Hz, H-11) and 7.21 (H-24), as well as two signals of the terminal
double bond at δ 4.98 (H-28a) and 4.96 (H-28b). These data
suggested that compound 6 is a 3,4-seco-triterpenoid derivative.18
The H and 13C NMR data were assigned by the H-1H COSY,
HMQC, and HMBC spectra (Tables 1 and 2). In the HMBC
spectrum, the trisubstituted olefinic proton at δ 5.58 showed long-
range correlations with C-12 (δ 37.9) and two quaternary carbons
1
1
Experimental Section
General Experimental Procedures. Melting points (uncorrected)
were measured on a Yanaco MP-S3 micromelting point apparatus.
Optical rotations were obtained with a JASCO DIP-370 polarimeter.
IR spectra were conducted on a JASCO FT/IR-300E spectrometer. 1D
1
(1H, 13C, and DEPT) and 2D (HMBC, HMQC, H-1H COSY, and
NOESY) NMR spectra were recorded on a JEOL ECP-500 NMR
spectrometer. The chemical shifts are quoted relative to TMS, and the
coupling constants are in Hz. FABMS and HRFABMS were taken on
a JEOL JMS-700 MStation. EIMS (70 eV) were obtained on a
Shimadzu GCMS-QP5050A spectrometer. The chromatographic silica
Figure 1. Main HMBC correlations of 1.

Lanostane-Type Triterpenoids from Kadsura coccinea
Journal of Natural Products, 2008, Vol. 71, No. 6 993
Chart 1
gel (200-300 mesh) was produced by Qingdao Ocean Chemical
Factory, and Sephadex LH-20 was obtained from GE Healthcare.
Plant Material. The roots of Kadsura coccinea were purchased from
Guangxi Medicinal Material Corporation, Nanning, China, and identi-
fied by Associate Professor Maoxiang Lai of Guangxi Institute of
Traditional Chinese Medicine. A voucher specimen (KC 0509) was
deposited in the Department of Natural Products Chemistry, Shenyang
Pharmaceutical University, Shenyang, People’s Republic of China.
Extraction and Isolation. The roots of K. coccinea (10 kg) were
extracted with 95% EtOH to give an alcohol extract. A portion (1340 g)
of the residue after removing EtOH was suspended in water and partitioned
with petroleum ether, CHCl3, EtOAc, and n-BuOH, successively. The
petroleum ether extract (150 g) was subjected to silica gel column
chromatography with a petroleum ether-EtOAc gradient solvent system
(100:0 f 0:100) to provide 12 fractions. Fractions 5 [petroleum
ether-EtOAc (100:7)], 6 [petroleum ether-EtOAc (100:8)], and 10
[petroleum ether-EtOAc (100:30)] were recrystallized using acetone to
afford compounds 1 (110 mg), 2 (290 mg), and 3 (10 mg), respectively.
Repeated chromatography of fraction 8 [petroleum ether-EtOAc (100:
15)] on a column of silica gel with gradient elution using petroleum ether
with increasing proportions of acetone (15:1 f 11:1) afforded compounds
4 (8 mg), 5 (7 mg), and 6 (6 mg). The CHCl3 extract (50 g) was fractionated
into 12 fractions by silica gel column chromatography. Fraction 8
[petroleum ether-acetone (100:15)] was subsequently chromatographed
on silica gel columns, together with Sephadex LH-20 [CHCl3-MeOH (1:
1)], to furnish compound 7 (22 mg).
Seco-coccinic acid B (2): colorless needles (acetone); mp 145-146
°C; [R]20 -25.4 (c 0.032, CHCl3); IR (KBr) νmax 2951, 2864, 1711,
D
1680, 1637, 1451, 1385, 1023, 957, 892 cm-1; 1H and 13C NMR data,
see Tables 1 and 2; HRFABMS m/z 455.3539 [M + H]+ (calcd for
C30H47O3, 455.3525).
Seco-coccinic acid C (3): colorless needles (acetone); mp 181-182
°C; [R]20 -28.2 (c 0.023, CHCl3); IR (KBr) νmax 3457, 2945, 2874,
D
1723, 1700, 1655, 1456, 1378, 1197, 1070, 976, 899 cm -1; H and
1
13C NMR data, see Tables 1 and 2; HRFABMS m/z 495.3438 [M +
Na]+ (calcd for C30H48O4Na, 495.3450).
Seco-coccinic acid D (4): colorless needles (acetone); mp 183-185
°C; [R]20 -31.1 (c 0.010, CHCl3); IR (KBr) νmax 3428, 2923, 2846,
D
2375, 1705, 1627, 1462, 1376, 911, 864, 818 cm-1; 1H and 13C NMR
data, see Tables 1 and 2; HRFABMS m/z 457.3639 [M + H]+ (calcd
for C30H49O3, 457.3682).
Seco-coccinic acid E (5): colorless needles (acetone); mp 161-162
°C; [R]20 -20.9 (c 0.020, CHCl3); IR (KBr) νmax 3264, 3070, 2958,
D
2923, 2874, 2650, 1696, 1455, 1375, 1315, 1273, 1106, 1068, 1003,
905 cm-1; 1H and 13C NMR data, see Tables 1 and 2; HRFABMS m/z:
479.3495 [M + Na]+ (calcd for C30H48O3Na, 479.3501).
Preparation of Methyl Ester of 5 (5a) and (R)- and (S)-MTPA
Esters of 5a (5b and 5c). After adding TMSCHN2 (0.2 mL) into a
solution of compound 5 (2.0 mg) in dried MeOH-THF (1:2, 1 mL),
the mixture was reacted for 10 h at room temperature to give the methyl
ester of compound 5 (5a, 2.3 mg). To each solution of compound 5a
(each 1.0 mg) in pyridine-d5 (0.75 mL) was separately added (R)-(-)-
MTPA Cl (10 L) and (S)-(+)-MTPA Cl (10 L) at room temperature,
followed by stirring at room temperature for 4 h. Each reaction mixture
Seco-coccinic acid A (1): colorless needles (acetone); mp 148-149
°C; [R]20 -36.8 (c 0.056, CHCl3); IR (KBr) νmax 2949, 2874, 1710,
D
1633, 1462, 1370, 1275, 1198, 1109, 1063, 902, 841 cm-1; 1H and 13
C
1
was transferred into a 5 mm NMR tube, and the H NMR data were
NMR data, see Tables 1 and 2; HRFABMS m/z 457.3674 [M + H]+
determined.
(calcd for C30H49O3, 457.3682).
(R)-MTPA Ester of 5a (5b): colorless, amorphous solid; 1H NMR
(pyridine-d5) δ 5.67 (1H, t, J ) 5.3 Hz, H-24), 5.28 (1H, d, J ) 2.9
Hz, H-7), 5.22 (1H, s, Ha-26), 5.03 (1H, s, Hb-26), 4.99 (1H, s, Ha-28),
4.97 (1H, s, Hb-28), 3.81 (3H, s, OMe), 2.60 (1H, m, H-9), 1.87 (1H,
m, Ha-23), 1.82 (3H, s, H-29), 1.77 (3H, s, H-27), 1.57 (1H, m, Hb-
23), 1.33 (1H, m, H-20), 1.00 (3H, s, H-30), 0.85 (3H, s, H-18), 0.81
(3H, d, J ) 6.2 Hz, H-21), 0.76 (3H, s, H-19).
1
(S)-MTPA Ester of 5a (5c): colorless, amorphous solid; H NMR
1
Figure 2. ∆δSR () δS - δR) values obtained from the H NMR
(pyridine-d5) δ 5.64 (1H, t, J ) 5.7 Hz, H-24), 5.28 (1H, brs, H-7),
5.11 (1H, s, Ha-26), 4.99 (1H, s, Hb-26), 4.99 (1H, s, Ha-28), 4.96 (1H,
spectra of 5b and 5c.

994 Journal of Natural Products, 2008, Vol. 71, No. 6
Wang et al.
s, Hb-28), 3.81 (3H, s, OMe), 2.62 (1H, m, H-9), 1.94 (1H, m, Ha-23),
1.83 (3H, s, H-29), 1.67 (3H, s, H-27), 1.65 (1H, m, Hb-23), 1.40 (1H,
m, H-20), 1.01 (3H, s, H-30), 0.88 (3H, d, J ) 6.2 Hz, H-21), 0.85
(3H, s, H-18), 0.78 (3H, s, H-19).
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Liu, J. S.; Huang, M. F.; Gao, Y. L. Acta Chim. Sin. 1980, 38, 361–
369.
(2) Chen, Y. G.; Hai, L. N.; Liao, X. R.; Qin, G. W.; Xie, Y. Y.;
Halaweish, F. J. Nat. Prod. 2004, 67, 875–877.
(3) Chen, Y. G.; Xie, Y. Y.; Cheng, K. F.; Cheung, K. K.; Qin, G. W.
Phytochemistry 2001, 58, 1277–1280.
Seco-coccinic acid F (6): colorless needles (acetone); mp 111-112
°C; [R]20 +51.8 (c 0.022, CHCl3); IR (KBr) νmax 2926, 2856, 1698,
D
1655, 1456, 1385, 1284, 1069, 897 cm-1; H and 13C NMR data, see
1
Tables 1 and 2; HRFABMS m/z 471.3472 [M + H]+ (calcd for
C30H47O4, 471.3474).
Coccinilactone A (7): colorless needles (acetone); mp 178-179 °C;
[R]20D +80.2 (c 0.027, CHCl3); IR (KBr) νmax 2946, 2874, 2377, 2345,
1715, 1464, 1368, 1292, 1209, 1118, 1045, 977 cm-1; 1H and 13C NMR
data, see Tables 1 and 2; HRFABMS m/z 457.3685 [M + H]+ (calcd
for C30H49O3, 457.3682).
(4) Li, L. N.; Xue, H. Planta Med. 1986, 52, 492–493.
(5) Li, L. N.; Xue, H.; Kangouri, K.; Kawashima, A.; Omura, S. Planta
Med. 1989, 55, 548–550.
(6) Dai, P.; Han, G. Q.; Arison, B. H. Chem. J. Chin. UniV. 1990, 11,
423–424.
(7) Kangouri, K.; Miyoshi, T.; Kawashima, A.; Ikeda, A.; Mizutani, T.;
Omura, S.; Li, L. N.; Xue, H. Planta Med. 1989, 55, 297–299.
(8) Li, L. N.; Xue, H.; Ge, D. L.; Kangouri, K.; Miyoshi, T.; Omura, S.
Planta Med. 1989, 55, 300–302.
(9) Tan, R.; Xue, H.; Li, L. N. Planta Med. 1991, 57, 87–88.
(10) Li, L. N.; Xue, H.; Kangouri, K.; Ikeda, A.; Omura, S. Planta Med.
1989, 55, 294–296.
Cell Culture and Growth Inhibition Assay. Human leukemia HL-
60 cells (obtained from American Type Culture Collection, Rockville,
MD) were cultured in RPMI-1640 medium (Gibco, New York, NY)
supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 1
mmol glutamine, and 10% heat-inactivated fetal bovine serum (Gibco).
Cell growth inhibition assay was performed as reported previously.20
Cells were seeded at a density of 1 × 105 cells/mL and incubated with
various concentrations of the tested compounds for 3 days. The
compounds were dissolved in dimethyl sulfoxide (DMSO), and the
amount of DMSO was controlled lower than 0.1% in the final
contration. The number of cells in each group was determined by
hemocytometer, and the cell viability was determined using trypan blue
staining. The growth inhibitory ability of these compounds was
calculated and expressed as the ratio of the cell number in treated group
to that of the untreated group. The concentration (GI50) that inhibited
half of the cell growth was calculated. 5-Fluorouracil (5-FU) was used
as a positive control, and 0.1% DMSO was used as a negative control.
(11) You, Z. P.; Liao, M. J.; Shi, Y. H.; Chen, Y. Z. Acta Pharm. Sin.
1997, 32, 455–457.
(12) Liu, J. S.; Huang, M. F. Acta Chim. Sin. 1991, 49, 502–506.
(13) Chen, D. F.; Zhang, S. X.; Wang, H. K.; Zhang, S. Y.; Sun, Q. Z.;
Cosentino, L. M.; Lee, K. H. J. Nat. Prod. 1999, 62, 94–97.
(14) Committee of Pharmacopoeia of People’s Republic of China. Phar-
macopoeia of People’s Republic of China; Chemical and Industrial
Publishing House: Beijing, 2005; Vol. 1, Suppl., p 24.
(15) L. R.; Hong, X.; Ding, X. L.; Zang, Z. Y. Dictionary of Modern Science
of Chinese Materia Medica; People’s Health Publishing House:
Beijing, 2001; Vol. 2, pp 2172-2173.
(16) Tai, T.; Akahori, A.; Shingu, T. Phytochemistry 1991, 30, 2796–2797.
(17) Chen, Y. G.; Qin, G. W.; Xie, Y. Y. Chem. Res. Appl. 2001, 13, 363–
367.
(18) Kim, H. J.; Choi, E. H.; Lee, I. S. Phytochemistry 2004, 65, 2545–
2549.
(19) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(20) Wang, F.; Hua, H. M.; Pei, Y. H.; Chen, D.; Jing, Y. K. J. Nat. Prod.
2006, 69, 807–810.
Acknowledgment. The authors wish to thank Assoc. Prof. M. Lai
(Guangxi Institute of Traditional Chinese Medicine, Nanning, People’s
Republic of China) for identification of the plant. This work was
partially supported by the Joint Research Fund for Overseas Chinese
Young Scholars of the National Natural Science Foundation of China
(30328030).
NP7007522