Pregnane, coumarin and lupane derivatives and cytotoxic constituents from Helicteres angustifolia

Article abstract of DOI:10.1016/j.phytochem.2006.03.005

2α,7β,20α-Trihydroxy-3β,21-dimethoxy-5-pregnene (1), 6,7,9α-trihydroxy-3,8,11α-trimethylcyclohexo-[d,e]-coumarin (2), 3β-hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid (3), and 3β-hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid methyl ester (4), along with 24 known compounds were isolated and structurally characterized from roots and aerial parts of Helicteres angustifolia (Sterculiaceae). In a preliminary bioassay, the two cucurbitacin derivatives, cucurbitacin D and J exhibited significant inhibitory activities against the growth of both hepatocellular carcinoma BEL-7402 cells and malignant melanoma SK-MEL-28 cells in vitro.

Full text of DOI:10.1016/j.phytochem.2006.03.005

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 1041–1047
www.elsevier.com/locate/phytochem
Pregnane, coumarin and lupane derivatives and cytotoxic
constituents from Helicteres angustifolia
a
a
a,
*
Wenliang Chen a,b, Weidong Tang , Liguang Lou , Weimin Zhao
a
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Department of Natural Products Chemistry,
555 Zuchongzhi Road, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China
b
Graduate School of the Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China
Received 26 July 2005; received in revised form 15 February 2006
Available online 24 April 2006
Abstract
2a,7b,20a-Trihydroxy-3b,21-dimethoxy-5-pregnene (1), 6,7,9a-trihydroxy-3,8,11a-trimethylcyclohexo-[d,e]-coumarin (2), 3b-
hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid (3), and 3b-hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid methyl ester (4), along
with 24 known compounds were isolated and structurally characterized from roots and aerial parts of Helicteres angustifolia (Sterculi-
aceae). In a preliminary bioassay, the two cucurbitacin derivatives, cucurbitacin D and J exhibited significant inhibitory activities against
the growth of both hepatocellular carcinoma BEL-7402 cells and malignant melanoma SK-MEL-28 cells in vitro.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Helicteres angustifolia; Sterculiaceae; Pregnane; Coumarin; Triterpenoid; Cytotoxicity
1. Introduction
cally. As a result, 28 compounds were isolated and eluci-
dated, of which four were new based on extensive NMR
analysis and chemical methods. In a preliminary bioassay,
the two cucurbitacin derivatives, cucurbitacin D and J were
found to exhibit significant inhibitory activities against the
growth of both hepatocellular carcinoma BEL-7402 cells
and malignant melanoma SK-MEL-28 cells.
The dried roots and stems of Helicteres angustifolia (L.)
(Sterculiaceae) are used in folk medicine as analgesic, anti-
inflammatory and anti-bacterial agents (Jiangsu New Med-
ical College, 1986) in the south of China under the name
Shan-Zhi-Ma; tumor growth inhibitory activity has also
been reported for this plant (Chiu and Chang, 1995). Pre-
vious phytochemical investigations of Helicteres plants
mainly led to identification of quinones derived from oxi-
dation of a cadinane-type sesquiterpenoid (Chen et al.,
1990) as well as triterpenoids with lupane and oleanane-
type skeletons (Liu and Wang, 1985; Chang et al., 2001).
In a continuation of our efforts to find new anti-tumor nat-
ural products from Chinese herb medicine, the constituents
of Helicteres angustifolia (L.) were investigated systemati-
2. Results and discussion
Compound 1 was obtained as white amorphous powder
with an elemental formula C23H38O5 as deduced by HR-
EIMS and NMR analyses. Its IR spectrum indicated the
existence of hydroxy (3425 cmꢀ1) and olefinic functional
(1630 cmꢀ1) groups in its structure. The 1H NMR spectrum
of 1 exhibited one singlet olefinic proton signal at dH 5.40,
two methoxyl resonances at dH 3.46 and 3.43, and two
singlet methyl signals at dH 1.12 and 0.77. Its 13C NMR
*
Corresponding author. Tel.: +86 21 50806052; fax: +86 21 50807088.
E-mail address: wmzhao@mail.shcnc.ac.cn (W. Zhao).
0031-9422/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.03.005

1042
W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
spectrum displayed 23 resonances, which through a DEPT
experiment were subclassified into four methyls (two for
methoxyls), seven methylenes (one oxygenated methylene),
nine methines (one for a sp2 methine and four for oxygen-
ated methines), and three quaternary carbons (one for a sp2
carbon). Analysis of the 1H–1H COSY spectrum of 1
revealed the following three fragments –C-1–C-2–C-3–C-
4–, –C-6–C-7–C-8–C-9–C-11–C-12–, and –C-14–C-15–C-
16–C-17–C-20–C-21– in its structure. The structure of 1
was established on the basis of analysis of the HMBC spec-
ser to H-17 than Hb-21, no matter whether the 20-OH
group was in an a or b orientation. Therefore, Ha-21 gave
a NOE correlation with H-17 (Fig. 2). The dihedral angles
between H-20 and Ha-21 were close to 180ꢁ and 45ꢁ, when
the 20-OH group was in an a or b orientation, respectively.
Therefore, the coupling constants between H-20 and Ha-
21, and between H-20 and Hb-21 could be used in the
determination of the relative configuration of 20-C of 1.
The relatively large coupling constant between H-20 and
Ha-21 (8.5 Hz), and the small coupling constant between
H-20 and Hb-21 (2.9 Hz) indicated that the 20-OH group
was in an a orientation (Fig. 3). According to the literature,
the configuration of the 20-OH group of pregnane deriva-
tive could also be determined by the magnitude of the
upfield shift of the 18-CH3 group of the 20-OH acylated
product. The principles used were the van der Waals effect
of the 20-OH group and the diamagnetic effect of the acyl
group to 18-CH3, which was stronger in the 20b-OH series
than in the 20a-OH series (Lee and Wolff, 1967). To con-
firm the configuration of C-20, 1 was peracetylated to give
1a, and little variance was observed between the chemical
shifts of CH3-18 of 1 and 1a (DdH = 0.01 ppm) thereby sug-
gesting an a-configuration of the 20-OH group in 1 (Naka-
tani et al., 1985). Therefore, the structure of 1 was
determined to be 2a,7b,20a-trihydroxy-3b,21-dimethoxy-
1
trum, in which H–13C long-range correlation signals were
observed as follows between: H-19 and C-1, C-5, C-9; H-18
and C-12, C-14, C-17; H-22 and C-3; H-23 and C-21; C-5
and H-1, H-7; H-6 and C-4, C-8, C-10, and C-14 and H-
7, H-9, H-12 (Fig. 1). In the NOESY spectrum of 1,
NOE correlations were observed at H-19/H-1b, H-19/H-
2, H-19/H-4b, H-3/H-4a, H-7/H-9, H-7/H-14, and H-18/
H-20 (Fig. 2). The relative configuration of 1 was thus
characterized completely, except for C-20. The relative con-
figuration at C-20 was subsequently deduced as follows:
since only one of the protons attached to C-21 showed a
NOE correlation signal to H-17, the analysis of the 3D-
structure of 1 generated from the molecular modeling
(CS Chem3D Ultra 9.0) using a MM2 force field calcula-
tion for energy minimization revealed that Ha-21 was clo-
HO
18
21
23
20
17
10
14
OH
O
11
9
12
11
19
13
12
HO
HO
16
1
5
8
4
4a
8a
HO
O
6
7
3
2
8
9
14
15
2
10
1
O
7
3
O
22
4
6
5
OH
13
2
1
Fig. 1. 1H–1H COSY (
) and key 1H–13C long-range correlation signals (
) in the HMBC spectra of 1 and 2.
23
OH
H
O
18
21
19
20
Ha
13
14
H
17
11
7
12
Hb
H
8
H
15
9
1
2
10
HO
OH
16
O
4
22
H
3
5
H
H
6
H
H
H
Fig. 2. Main NOE signals (
) observed in the NOESY spectrum of 1.

W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
1043
NMR spectrum of 3 was similar to that of the known com-
pound helicteric acid (5) isolated from the same plant
(Chang et al., 2001), except for differences in chemical
shifts at C-2 (+3.8 ppm), C-3 (ꢀ1.9 ppm), and C-4
(+1.0 ppm) (see Table 2), i.e. due to the absence of the ace-
tyl group at 3-OH. Acetylation of 3 with acetic anhydride
in pyridine yielded 5 as the sole product. Therefore, com-
pound 3 was characterized to be 3b-hydroxy-27-benzoyl-
oxylup-20(29)-en-28-oic acid.
OMe
OMe
HO
Hb
H(20)
Ha
(20)H
Hb
OH
Ha
C(17)
C(17)
20-epimer of 1
J (H-20, Ha-21) = 2.9 Hz
J (H-20, Hb-21) = 8.5 Hz
1
J (H-20, Ha-21) = 8.5 Hz
J (H-20, Hb-21) = 2.9 Hz
Compound 4 was obtained as white amorphous powder,
and its molecular formula was deduced as C38H54O5 by
HR-EIMS and NMR spectroscopic analyses. Its NMR
spectra were superimposed with those of 3 except for the
up-field shift of C-28 (ꢀ3.9 ppm), and additional reso-
nances for one methoxyl group, suggesting that 4 was the
methyl ester of 3. Therefore, the structure of 4 was deter-
mined to be 3b-hydroxy-27-benzoyloxylup-20(29)-en-28-
oic acid methyl ester. To the best of our knowledge, 3
and 4 are new compounds.
Fig. 3. Newman projection diagrams of C(20)–C(21) in 1 (20a-OH) and
its epimer (20b-OH).
5-pregnene. To the best of our knowledge, compound 1 is a
new compound.
Compound 2 was obtained as brown amorphous pow-
der with an elemental formula C15H16O5 as also deduced
by HR-EIMS and NMR analyses. The maxima absorption
at 320 nm in the UV spectrum revealed a conjugated sys-
tem in its structure, and the IR spectrum indicated the exis-
tence of hydroxy (3398 cmꢀ1), conjugated carbonyl
(1664 cmꢀ1), and olefinic (1614 cmꢀ1) functionalities. In
the 13C NMR spectrum, the 15 carbon signals consisted
of three methyls, one methylene, two methines, and nine
quaternary carbons (one for an ester carbonyl and the
Besides the four new natural products, 24 other known
compounds were also isolated from Helicteres angustifolia,
and their structures were characterized by comparison of
the physical and NMR data with those reported in litera-
ture, or co-TLC with authentic samples as helicteric acid
(5) (Chang et al., 2001), methyl helicterate (6) (Chang
et al., 2001), 3b-acetoxyl-27-(p-hydroxylbenzoyloxy)-lup-
20(29)-en-28-oic acid methyl ester (7) (Chang et al.,
2001), 3b-acetoxylup-20(29)-en-28-ol (8) (Hiroya et al.,
2002), 3b-hydroxylup-20(29)-en-28-oic acid 3-caffeate (9)
(Pan et al., 1994), betulinic acid (10), cucurbitacin D (11)
(Velde and Lavie, 1983), cucurbitacin J (12) (Kanchana-
poom et al., 2002), oleanolic acid (13), b-sitosterol (14),
mansonone E (15) (Chen et al., 1990), mansonone F (16)
(Chen et al., 1990), mansonone H (17) (Chen et al.,
1990), mansonone H methyl ester (18) (Chen et al.,
1990), lariciresinol (19) (Fonseca et al., 1978), dihydrode-
hydrodiconiferyl alcohol (20) (Fukuyama et al., 1996),
(+)-pinoresinol (21) (Ludwig et al., 1964), lirioresinol-B
(22) (Tatematsu et al., 1984), rosmarinic acid (23) (Satake
et al., 1999), 8-O-b-D-glucuronyl-hypolaetin 40-methyl
ether (24) (Billeter et al., 1991), coniferyl alcohol (25)
(Steeves et al., 2001), 3-(3,4-dimethoxyphenyl)-2-propenal
(26) (Ito et al., 2001), kaempferol 3-O-b-D-glucopyranoside
(27) (Beninger and Hosfield, 1998), 5,8-dihydroxy-7,40-
dimethoxyflavone (28) (Ramesh and Yuvarajan, 1995).
Among them, 8, 9, 11, 12, 19-22, and 25-27 were reported
for the first time from the genus Helicteres.
1
other eight for sp2 carbons). The H NMR spectrum of 2
exhibited two singlet methyl signals at dH 2.18 and 2.10,
and one doublet methyl resonance at dH 1.36 (3H, d,
J = 7.3 Hz). A structural fragment –C-9–C-10–C-11–C-
1
14– could be deduced from the H–1H COSY spectrum
of 2. The structure of 2 was further established on the basis
of analysis of its HMBC spectrum (Fig. 1), in which the
1H–13C long-range correlation signals were observed
between: H-12 and C-2, C-3, C-4; H-13 and C-7, C-8, C-
8a; H-9 and C-4, C-4a; H-11 and C-4a, C-5, C-6, and H-
14 and C-5, respectively. The relative configurations of
C-9 and C-11 were determined according to NOE correla-
tions between H-9 and H-11, between H-11 and H-10b,
and between H-14 and H-10a in its NOESY spectrum.
The structure of 2 was therefore established as 6,7,9a-trihy-
droxy-3,8,11a-trimethylcyclohexo-[d,e]-coumarin. It was a
highly oxidized derivative possessing cadinane-type skele-
ton (Wang et al., 1988), and to the best of our knowledge,
it is a new compound.
Compound 3 was obtained as a white amorphous pow-
der with a molecular formula C37H52O5 as also deduced by
1
the HR-EIMS and NMR analyses. Its H NMR spectrum
revealed the existence of one mono-substituted phenyl, one
terminal methylene, one oxygenated methylene, one oxy-
genated methine, and five single methyl groups (Table 2).
The 13C NMR spectrum of 3 displayed 37 signals which
following a DEPT analysis were separated into five methyl,
twelve methylene (one for a sp2 methylene and one for an
oxygenated methylene), eleven methine (one for an oxygen-
ated methine and five for sp2 methines), and nine quater-
nary carbon (two for ester/carboxylic carbonyl, and two
for sp2 carbons) functionalities, respectively. The 13C
Helicteres angustifolia was reported to exhibit tumor
growth inhibitory activity (Chiu and Chang, 1995).
According to the literature, cucurbitacins possessed signif-
icant anti-tumor activity (Gitter et al., 1961; Gallily et al.,
1962; Duncan et al., 1996), and betulinic acid was found
to be a selective inhibitor of human melanoma functioned
by induction of apoptosis (Pisha et al., 1995). In order to
identify the anti-tumor constituents of H. angustifolia,
compounds 3–12 and 15–18 were evaluated against the
growth of human tumor cell lines, including hepatocellular

1044
W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
29
20
RO
18
23
21
20
O
30
H
12
21
17
19
13
19
11
H
12
26
18
16
15
22
H
9
14
RO
O
11
9
1
4
25
13
17
R3
2
3
10
8
H
H
14
H
16
7
5
15
22
6
1
4
8
2
3
10
OR
H
6
27
5
7
R2
1
R = H
1a R = acetyl
R1O
H
23
24
14
3 R1 = H, R2 = benzoyloxy, R3 = 2C8OOH
4 R1 = H, R2 = benzoyloxy, R3 = COOCH3
5 R1 = acetyl, R2 = benzoyloxy, R3 = COOH
6 R1 = acetyl, R2 = benzoyloxy, R3 = COOCH3
7 R1 = acetyl, R2 = 4-hydoxylbenzoyloxy, R3 = COOCH3
8 R1 = acetyl, R2 = H, R3 = CH2OH
OH
10
9
11
HO
HO
12
4
5
8
6
4a
3
2
7
1
8a
O
O
9 R1 = cafeoyl, R2 = H, R3 = COOH
10 R1 = R2 = H, R3 = COOH
13
2
O
O
OH
OH
OH
H
H
O
H
O
H
H
OH
H
OH
HO
O
OH
OH
HO
O
12
11
carcinoma BEL-7402 cell line and malignant melanoma
SK-MEL-28 cell line. It was found that the two cucurbita-
cin derivatives, 11 and 12, significantly inhibited the prolif-
eration of BEL-7402 cells (with IC50s of 1.41 and 1.37 lM,
respectively) and SK-MEL-28 cells (with IC50s of 1.22 and
1.28 lM, respectively). The three betulinic acid derivatives
3, 5 and 9 possessed only mild inhibitory activity against
SK-MEL-28 cells with IC50s of 20.5, 81.7 and 32.0 lM,
respectively. These cytotoxic components found from H.
angustifolia may contribute in part to the tumor growth
inhibitory activity of this medicinal plant.
Finnigan LCQ-DECA instrument, LR-EIMS were
obtained on a MAT-95 spectrometer, and HR-EIMS
were obtained on Kratos 1H spectrometer. NMR spectra
were acquired on a Bruker AM 400 spectrometer with
TMS as internal standard. CC graphic separations were
carried out using a LiChroprep RP-18 Lobar column
(40–63 lm, Merck), silica gel H60 (300–400 mesh, Qingdao
Haiyang Chemical Group Corporation, Qingdao, People’s
public of China), MCI Gel CHP20P (75–150 lm; Mitsubi-
shi Chemical Industries) and Sephadex LH-20 (Pharmcia
Biotech AB, Uppsala, Sweden), respectively. HSGF254
silica gel TLC plates (Yantai Chemical Industrial Institute,
Yantai, People’s Republic of China) and RP-18 WF254s
TLC plates (Merck) were used for analytical TLC.
3. Experimental
3.1. General
3.2. Plant material
Optical rotations were measured with Perkin–Elmer
241MC polarimeter or Perkin–Elmer 341 polarimeter.
UV spectra were recorded with a Beckman DU-7 spec-
trometer. IR spectra were recorded using a Perkin–Elmer
577 spectrometer. LR-ESIMS was measured using a
Whole plants of Helicteres angustifolia (L.) were col-
lected in the suburb of Guangzhou, Guangdong Province,
P.R. China, in May 2003, and identified by Professor Zex-
ian Li of the South China Institute of Botany, Chinese
Academy of Sciences.
A
voucher specimen (No.

W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
1045
SIMMS0305) is deposited in the herbarium of Shanghai
Institute of Materia Medica, Chinese Academy of Sciences.
give Fr.B2A (800 mg), Fr.B2B (648 mg) and Fr.B2C
(351 mg), respectively. Fr.B2A was separated by prepara-
tive TLC developed with CHCl3–EtOAc (5:1), to afford
28 (3 mg). Fr.B2B was successively subjected to Sephadex
LH-20 CC eluted with EtOH–H2O (95:5), and silica gel
CC eluted with CHCl3–MeOH–H2O (4:1:0.1), to yield 24
(80 mg) and 27 (4 mg). Fr.B2C was separated by prepara-
tive TLC developed with CHCl3–MeOH–H2O (4:1:0.1),
to give 23 (5 mg); Fr.B3 was purified by a RP-18 column
eluted with MeOH–H2O (1:1) to afford 2 (40 mg).
3.3. Extract and isolation
Powdered air-dried roots of Helicteres angustifolia (L.)
(500 g) were extracted with boiling methanol thrice
(1.5 L · 3, each time for 2 hours). After evaporation of
the methanol in vacuo, the residue was suspended into
1.0 L water and then extracted with CHCl3 and nBuOH
(both 500 mL · 3) successively, to afford CHCl3 (8 g) and
n-BuOH fraction (40 g) solubles. The chloroform solubles
were subjected to silica gel CC, eluted with a gradient of
petroleum ether–EtOAc (10:1–0:1) to give Fr.C1
(902 mg), Fr.C2 (1.34 g), Fr.C3 (1.91 g), Fr.C4 (943 mg)
and Fr.C5 (2.53 g). Fr.C1 was purified by silica gel CC
eluted with petroleum ether–acetone (40:1) to give 6
(87 mg); Fr.C2 was subjected to repeated column chroma-
tography over silica gel using petroleum ether–acetone
(25:1) as eluent to yield 5 (400 mg), 8 (22 mg), and 14
(68 mg); Fr.C3 was also subjected to silica gel CC eluted
with petroleum ether–acetone (20:1) afforded 3 (10 mg), 4
(19 mg), 7 (19 mg), 10 (12 mg), and 13 (10 mg); 9 (25 mg)
was obtained from Fr.C4 by silica gel CC, eluted with
petroleum ether–acetone (10:1); Fr.C5 was also subjected
to repeated silica gel CC using petroleum ether–acetone
(8:1) as eluent to give 15 (20 mg), 16 (40 mg), 17 (10 mg),
and 18 (5 mg).
3.4. 2a,7b,20a-Trihydroxy-3b,21-dimethoxy-5-pregnene (1)
20
White amorphous powder, ½aꢁD : þ6:1 (c 0.43, CHCl3);
IR (KBr): mmax cmꢀ1: 3425, 2937, 1689, 1630, 1458, 1385,
1194, 1099, 1022, 731; EIMS (probe) 70 eV, m/z (rel. int.)
394 [M]+ (9), 376 (44) 362 (100), 149 (54), 86 (54); HR-
EIMS, m/z: 394.2711 [M]+ (Calcd for C23H38O5,
1
394.2719); for H and 13C NMR spectroscopic data, see
Table 1.
3.5. Acetylation of compound 1
Compound 1 (10 mg) and Ac2O (0.2 mL) were added to
pyridine (2 mL), with the resulting mixture stirred at 80 ꢁC
Table 1
Powdered air-dried aerial parts of Helicteres angustifolia
(L.) (3.0 kg) were percolated at room temperature with
EtOH–H2O (95:5, 5.0 L · 3). The filtrate was concentrated
to dryness in vacuo and then suspended in EtOH–H2O
(1:4) overnight. After filtration of the precipitated chloro-
phyll and evaporation of the EtOH in the filtrate, the aque-
ous residue (1.0 L) was extracted with CHCl3 and n-BuOH
(both 500 mL · 3), successively, to yield CHCl3 (10 g) and
n-BuOH (25 g) solubles. The chloroform solubles were sub-
jected to silica gel CC, eluted with a gradient of petroleum
ether–EtOAc (10:1–0:1) to give Fr.C01 (3.11 g), Fr.C02
(1.7 g) and Fr.C03 (4.6 g). Fr.C01 was subjected to repeated
silica gel CC, using petroleum ether–EtOAc (5:1) as eluent,
to afford 26 (3 mg), 19 (28 mg), 21 (28 mg) and 25 (10 mg);
Fr.C02 was separated by a silica gel CC, eluted with petro-
leum ether–EtOAc (2:1), and then subjected to preparative
TLC developed with Et2O to afford 1 (67 mg), 11 (34 mg),
and 12 (5 mg); Fr.C03 was applied to silica gel column
which was eluted with a gradient of CHCl3–acetone
(20:1–1:1) to give Fr.C03 A (300 mg), Fr.C03B (310 mg)
and Fr.C03 C (3.6 g). 22 (36 mg) was obtained from
Fr.C03A by Sephadex LH-20 CC eluted with EtOH–H2O
(95:5), while 20 (43 mg) was obtained from Fr.C03B by a
RP-18 column eluted with MeOH–H2O (35:65). The
n-BuOH fraction was separated by a MCI column succes-
sively, eluted with H2O, MeOH–H2O (1:1) and MeOH–
H2O (9:1), to give Fr.B1 (20 g), Fr.B2 (2.0 g) and Fr.B3
(300 mg), respectively. Fr.B2 was subjected to silica gel
CC eluted with a gradient of CHCl3–MeOH (10:1–1:1) to
1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of 1 (CDCl3)
and 2 (CD3OD) (J in Hz)
No.
1
2
13C NMR 1H NMR
13C NMR 1H NMR
1
44.0 t
1.11, m (a); 2.14, dd,
4.8, 12.7 (b)
2
3
4
70.5 d
85.0 d
34.8 t
3.75, m
2.97, m
2.20, dd, 2.2, 11.5 (a);
2.56, dd, 4.9, 11.5 (b)
166.5 s
119.2 s
149.5 s
4a
5
6
7
8
109.2 s
126.2 s
140.8 s
147.7 s
111.0 s
149.3 s
142.0 s
126.3 d
73.1 d
40.1 d
5.40, br s
3.92, br d, 7.8
1.44, m
8a
9
48.4 d
37.9 s
26.4 t
38.3 t
52.2 s
55.4 d
20.8 t
24.8 t
42.2 d
12.8 q
20.2 q
71.1 d
76.5 t
1.17, m
65.2 d
37.1 t
27.4 d
4.98, br s
1.90, m; 2.11, m
3.32, m
10
11
12
13
14
15
16
17
18
19
20
21
1.95, m
1.17, m (a); 1.82, m (b) 12.5 q
8.9 q
2.10, s
2.18, s
1.25, m
22.6 q
1.36, d, 7.3
1.63, m
1.95, m
1.44, m
0.77, s
1.12, s
3.80, m
3.28, dd, 8.5, 9.3;
3.54, dd, 2.9, 9.3
3.46, s
22
23
56.6 q
58.9 q
3.43, s

1046
W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
Table 2
for 5 h. Further cooling to room temperatures, as evapora-
tion of the solvent to dryness in vacuo, the residue was
subjected to preparative TLC developed with petroleum
ether–CHCl3 (1:6) to afford 1a (7 mg). 2a,7b,20a-triacet-
oxy-3b,20-dimethoxy-5-pregnene (1a): white amorphous
1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of 3 and 4
(CDCl3) (J in Hz)
No.
3
4
13C NMR 1H NMR
13C NMR 1H NMR
20
powder, ½aꢁD : ꢀ25:6 (c 0.348, CHCl3); ESI-MS, m/z: 543
1
2
3
38.9 t
27.4 t
38.9 t
27.4 t
[M+Na]+, 1063 [2M+Na]+; 1H NMR (400 MHz, in
CDCl3): d 5.32 (1H, br s, H-6), 5.12 (1H, m, H-20), 5.06
(1H, br d, 7.8, H-7), 5.02 (1H, m, H-2), 3.50 (1H, dd, 8.5,
9.3, H-21), 3.44 (1H, dd, 2.9, 9.3, H-21), 3.41 (3H, s, 21-
OCH3), 3.37 (3H, s, 3-OCH3), 3.17 (1H, m, H-3), 2.10,
2.08 and 2.05 (each 3H, s, 3 · -COCH3), 1.17 (3H, s, 19-
CH3), 0.76 (3H, s, 18-CH3).
78.8 d
3.20, dd,
78.7 d
3.15, dd,
5.2, 10.8
5.3, 11.1
4
5
6
7
8
9
38.8 s
55.5 d
18.3 t
35.4 t
41.6 s
52.1 d
37.6 s
21.1 t
25.4 t
39.3 d
45.8 s
24.4 t
32.7 t
56.2 s
49.7 d
46.9 d
150.1 s
30.5 t
36.7 t
27.9 q
16.7 q
16.5 q
15.4 q
63.9 t
38.8 s
55.5 d
18.3 t
35.4 t
41.5 s
52.1 d
37.5 s
21.1 t
25.4 t
39.2 d
45.7 s
24.3 t
32.6 t
56.4 s
49.9 d
46.9 d
150.1 s
30.5 t
36.6 t
27.9 q
16.7 q
16.4 q
15.4 q
63.9 t
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
3.6. 6,7,9a-Trihydroxy-3,8,11a-trimethylcyclohexo-[d,e]-
coumarin (2)
20
Brown amorphous powder, ½aꢁD : þ30 (c 1.17, MeOH);
UV (MeOH) kmax nm (log e): 320 (3.6), 209 (4.3); IR (KBr):
mmax cmꢀ1: 3398, 2929, 1664, 1614, 1574, 1387, 1244, 1192,
1132, 1016, 970, 775; EIMS, m/z (rel. int.) 276 [M]+ (100),
261 (28), 243 (60), 231 (23), 215 (27); HR-EIMS, m/z:
276.0989 [M]+ (Calcd. for C15H16O5, 276.0998); for 1H
and 13C NMR spectroscopic data, see Table 1.
3.06, m
3.02, m
0.74, s
1.03, s
0.91, s
0.87, s
4.54, d, 12.9;
4.78, d, 12.9
0.73, s
0.99, s
0.89, s
0.85, s
4.50, d, 12.9;
4.67, d, 12.9
3.7. 3b-Hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid
(3)
20
White amorphous powder, ½aꢁD : ꢀ2:2 (c 0.31, CHCl3);
UV (MeOH) kmax nm (loge): 230 (4.2), 275 (w); IR
(KBr): mmax cmꢀ1: 3429, 3070, 2945, 2872, 1716, 1641,
1603, 1452, 1377, 1315, 1273, 1176, 1117, 1070, 1026,
984, 885, 712; EIMS, m/z (rel. int.): 576 [M]+ (4), 558
[MꢀH2O]+ (7), 454 (74), 436 (55), 246 (38), 234 (26), 207
(95), 189 (80), 175 (26), 135 (41), 105 (100); HR-EIMS,
m/z: 576.3823 [M]+ (Calcd. for C37H52O5, 576.3815); for
1H and 13C NMR spectroscopic data, see Table 2.
28
29
180.5 s
110.0 t
176.6 s
109.9 t
4.63, s; 4.75, s
1.72, s
4.60, d, 1.8;
4.67, d, 1.8
1.68, s
30
19.5 q
19.5 q
51.4 q
28-COOCH3
3.66, s
Benzoyl moiety
10
20, 60
130.6 s
129.5 d
130.6 s
8.03, dd, 1.6, 7.2 129.5 d
8.00, dd,
1.6, 7.2
7.45, m
7.56, m
30, 50
40
70
128.5 d
133.0 d
166.8 s
7.47, m
7.58, m
128.5 d
133.0 d
166.7 s
3.8. 3b-Hydroxy-27-benzoyloxylup-20(29)-en-28-oic acid
methyl ester (4)
20
White amorphous powder, ½aꢁD : ꢀ5:8 (c 0.38, CHCl3);
UV (MeOH) kmax nm (loge): 230 (4.2), 275 (w); IR
(KBr): mmax cmꢀ1: 3535, 3070, 2949, 2872, 1718, 1643,
1603, 1452, 1377, 1315, 1271, 1165, 1117, 1070, 1026,
974, 885, 712; EIMS, m/z (rel. int.): 590 [M]+ (2), 572
[MꢀH2O]+ (4), 468 (95), 260 (38), 207 (80), 187 (72), 105
(100); HR-EIMS, m/z: 590.3981 [M]+ (Calcd. for
3.10. Determination of cell proliferation by MTT assay
Cells (5 · 104 cells/ml) were incubated with different
concentrations of compounds 3–12 and 15–18 for 72 h. Cell
proliferation was determined by MTT assay (Vistica et al.,
1991). The assays were carried out by triplicate in at least
three independent experiments.
1
C38H54O5, 590.3971); for H and 13C NMR spectroscopic
data, see Table 2.
3.9. Acetylation of compound 3
Acknowledgement
Acetylation of 3 (1 mg) was carried out as above.
The product was identified to be identical with 5 by
co-TLC analysis (developed with petroleum ether–ace-
tone 6:1).
The authors are grateful to Shanghai Committee of Sci-
ence and Technology for partial financial support (No.
03DZ19228).

W. Chen et al. / Phytochemistry 67 (2006) 1041–1047
1047
Lee, H., Wolff, M.E., 1967. C-18 functional steroids and D-homo steroids.
The Journal of Organic Chemistry 32, 192–196.
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