Cucurbitane-type triterpenoids from the stems of Cucumis melo

Article abstract of DOI:10.1021/np800692t

Phytochemical investigation of the stems of Cucumis melo led to the isolation and identification of 21 cucurbitane-type triterpenoids, including nine new compounds (1-9) and 12 known compounds. Their structures were determined on the basis of spectroscopi

Full text of DOI:10.1021/np800692t

824
J. Nat. Prod. 2009, 72, 824–829
Cucurbitane-Type Triterpenoids from the Stems of Cucumis melo
Chuan Chen,§ Shigao Qiang,§ Liguang Lou,* and Weimin Zhao*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai 201203, People’s Republic of China
ReceiVed October 29, 2008
Phytochemical investigation of the stems of Cucumis melo led to the isolation and identification of 21 cucurbitane-type
triterpenoids, including nine new compounds (1-9) and 12 known compounds. Their structures were determined on
the basis of spectroscopic analyses, chemical methods, and comparison with spectroscopic data in the literature. Two
known compounds, cucurbitacin B (10) and cucurbitacin A (11), showed significant cytotoxic activity against the
proliferation of A549/ATCC and BEL7402 cells in Vitro. Of the new compounds, only compound 7 was weakly cytotoxic.
The inhibitory effects of all compounds on the Jak-Stat3 signaling pathway were evaluated, but only cucurbitacin B
(10) showed significant inhibitory activity of phosphotyrosine STAT3.
Stems of Cucumis melo Linn (Cucurbitaceae) have been used
in traditional Chinese medicine for the treatment of dyspepsia,
jaundice, acute and chronic hepatitis, hepatic cirrhosis, hepatoma,
and cancer.1,2 Previous phytochemical studies have revealed C. melo
to be a rich source of volatile compounds,3 triterpenoids,4-11
sterols,12 and flavonoids.13 Cucurbitacins are noted for their
cytotoxicity and potential anticancer activity.14-19 Several published
studies have pointed out that the mechanism of activity of
cucurbitacins involves their interference with the Jaks-Stat (Janus
kinase-signal transducer and activator of transcription) signaling
pathway, specifically with the STAT3 signaling pathway.20-23
Cucurbitacins also exhibit other in Vitro or in ViVo pharmacological
effects, such as hepatoprotective, cardiovascular, purgative, anti-
inflammatory, antimicrobial, anthelmintic, and CNS effects and
antifertility activities.24-27 In our search for new anticancer agents
and JAK/STAT3 signaling pathway inhibitors from natural re-
sources, 21 cucurbitane-type triterpenoids, including nine new
compounds (1-9) and 12 known compounds, cucurbitacin B (10),28
23,24-dihydrocucurbitacin B,29 cucurbitacin A (11),30 cucurbitacin
R,31 isocucurbitacin R,32 cucurbitacin G,33 cucurbitacin H,33
hexanorcucurbitacin D,33 arvenin I,34 arvenin III,34 dihydroisocu-
curbitacin B,35 and 19-norlanosta-5, 24-dien-11-one,36 were isolated
from the stems of C. melo.
Results and Discussion
Powdered, air-dried stems of C. melo (5 kg) collected in Anhui
1
sp2 carbons, and one oxygenated carbon). The H and 13C NMR
Province, People’s Republic of China, were percolated at room
data of 1 (Tables 1, 2) were characteristic of the cucurbitacin
temperature with 95% ethanol three times. After evaporation of
structure with an additional ring formed by cyclization of the side
the ethanol in Vacuo, the aqueous residue was extracted successively
chain through an ether linkage according to the number of double-
with petroleum ether, CHCl3, ethyl acetate, and 1-butanol. The latter
bond equivalents.37 Analyses of its 1H-1H COSY and HSQC
three extracts were subjected to a series of chromatography steps
spectra led to the fragments C-10-C-1-C-2-C-3; C-6-C-7;
to afford 21 compounds.
C-15-C-16-C-17; and C-22-C-23-C-24. The chemical shifts of
Compound 1 was obtained as a white, amorphous powder with
C-2 and C-3 and the coupling constant between H-2 and H-3
the molecular formula C30H46O7, as deduced from HRESIMS and
suggested a 2,3-cis-diol structure on ring A.28,38-41 The HMBC
1
NMR analyses. Its H NMR spectrum revealed the existence of
spectrum indicated that the proton signal at δH 4.02 was interrelated
two olefinic protons (δH 6.22, 1H, d, 7.3 Hz and δH 5.85, 1H, d,
with C-5 (δC 145.2), C-6 (δC 123.3), C-9 (δC 49.5), and C-14 (δC
5.2 Hz), seven tertiary methyl groups (δH 0.97, 1.03, 1.11, 1.23,
48.9). The above results indicated that an OH was attached to C-7.
1.25, 1.28, 1.70), and seven protons bonded to carbons bearing
Furthermore, a 13C-1H long-range correlation signal between H-16
oxygen. The 13C NMR spectrum displayed 30 signals separated by
and C-23 revealed that C-16 and C-23 were linked via an ether
DEPT experiments into seven methyl, five methylene (one oxygen-
bond to form a pyranoid structural element. 13C-1H long-range
ated methylene), 10 methine (two sp2 methines and five oxygenated
correlation signals at H-23/C-25 and H-26/C-24 placed the olefinic
methines), and eight quaternary carbons (one carbonyl carbon, two
bond between C-24 and C-25, and an OH on one of the terminal
methyl groups. The NOE cross-peaks at H-24/H-16 and CH3-30/
* To whom correspondence should be addressed. Tel & Fax: +86-21-
H-17 indicated that H-23, H-17 was R-oriented. NOE cross-peaks
50806052. E-mail: wmzhao@mail.shcnc.ac.cn (W.Z., chemistry). Tel & Fax:
at H-2/H-10, H-3/H-1R, CH3-30/H-7, CH3-18/H-16, and CH3-21/
H-23 in the ROESY spectrum revealed that OH-2, OH-3, OH-7,
+86-21-50806056. E-mail: lglou@mail.shcnc.ac.cn (L.L., biology).
§ These authors contributed equally to this work.
10.1021/np800692t CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/06/2009

Cucurbitane-Type Triterpenoids from Cucumis melo
Journal of Natural Products, 2009, Vol. 72, No. 5 825

826 Journal of Natural Products, 2009, Vol. 72, No. 5
Chen et al.
Table 2. 13C NMR Data (δ) of Compounds 1-9 (100 MHz; 1, 2, 4, and 9 in CD3OD and others in CDCl3)
position
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
30.1 t
69.3 d
80.4 d
43.6 s
145.2 s
123.3 d
67.8 d
53.8 d
49.5 s
36.2 d
216.4 s
49.8 t
28.6 t
76.9 d
77.2 d
43.2 s
145.1 s
123.2 d
67.8 d
53.8 d
49.5 s
36.0 d
216.4 s
50.3 t
35.9 t
71.5 d
213.0 s
50.0 s
140.3 s
120.4 d
23.8 t
42.6 d
48.5 s
33.7 d
212.7 s
48.4 t
35.4 t
80.3 d
213.5 s
52.8 s
141.9 s
122.8 d
24.9 t
35.1 d
55.1 s
34.9 d
214.4 s
51.6 t
34.9 t
71.6 d
212.0 s
50.0 s
140.0 s
121.4 d
23.3 t
33.0 d
53.8 s
32.8 d
212.4 s
48.4 t
35.1 t
71.8 d
212.6 s
50.1 s
139.9 s
121.4 d
23.6 t
33.2 d
53.8 s
33.0 d
212.3 s
48.6 t
35.2 t
71.4 d
212.2 s
50.2 s
145.0 s
121.8 d
66.2 d
51.8 d
47.5 s
34.6 d
211.8 s
48.6 t
35.2 t
71.4 d
212.2 s
50.2 s
145.0 s
121.8 d
66.2 d
51.7 d
47.5 s
34.6 d
211.8 s
48.6 t
36.4 t
80.0 d
213.8 s
53.0 s
142.2 s
121.6 d
25.4 t
44.8 d
50.6 s
35.4 d
214.5 s
48.6 t
10
11
12
13
14
15
49.0 s
48.9 s
42.4 t
49.0 s
48.8 s
42.4 t
48.1 s
50.2 s
44.4 t
52.0 s
49.8 s
47.3 t
50.7 s
47.7 s
45.3 t
50.9 s
47.9 s
45.4 t
49.7 s
47.0 s
45.2 t
49.6 s
47.3 s
45.4 t
51.0 s
51.8 s
46.6 t
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
72.1 d
56.9 d
20.6 q
22.3 q
73.8 s
30.3 q
47.1 t
73.0 d
127.6 d
138.9 s
69.1 t
72.1 d
57.0 d
20.6 q
22.1 q
73.9 s
30.3 q
47.0 t
73.0 d
127.5 d
139.0 s
69.1 t
72.3 d
58.2 d
20.2 q
21.2 q
75.7 s
27.6 q
49.6 t
65.5 d
126.8 d
136.9 s
67.1 t
72.3 d
61.7 d
20.3 q
61.7 t
71.3 d
57.2 d
18.7 q
60.7 t
71.1 d
57.9 d
18.6 q
60.5 t
79.2 s
24.5 q
215.4 s
30.8 t
71.0 d
58.1 d
19.9 q
21.4 q
78.2 s
23.9 q
202.4 s
120.2 d
151.9 d
79.3 s
25.9 q
26.3 q
29.8 q
21.2 q
19.1 q
21.9 q
170.4 s
70.6 d
57.7 d
19.8 q
21.4 q
78.8 s
24.4 q
213.8 s
30.6 t
73.0 d
68.4 d
20.8 q
20.7 q
211.1 s
32.3 q
80.8 s
26.1 q
206.0 s
123.1 d
151.9 d
81.6 s
27.0 q
27.3 q
30.2 q
29.7 q
22.4 q
22.3 q
172.4 s
104.8 d
75.9 d
78.7 d
71.9 d
78.4 d
63.4 t
77.9 s
23.7 q
202.3 s
119.0 d
155.6 d
70.9 s
28.6 q
29.1 q
29.3 q
21.0 q
19.4 q
36.9 t
34.6 t
70.3 s
28.7 q
29.3 q
29.9 q
21.2 q
19.3 q
81.3 s
25.8 q
26.1 q
29.8 q
21.2 q
19.0 q
22.4 q
170.5 s
14.3 q
28.3 q
26.5 q
22.2 q
14.3 q
28.2 q
26.7 q
22.1 q
14.1 q
29.4 q
20.0 q
18.8 q
29.8 q
22.3 q
19.9 q
-OCOCH3
-OCOCH3
1′
2′
3′
4′
5′
6′
102.4 d
75.6 d
78.7 d
72.1 d
78.3 d
63.3 t
104.8 d
75.9 d
78.7 d
71.9 d
78.4 d
63.4 t
H-16, and OH-20 were ꢀ-oriented. The NOE correlation between
CH2-26 and H-24 revealed the E configuration of the olefinic bond.
Therefore, 1 was characterized as 16R,23R-epoxy-2ꢀ,3ꢀ,7ꢀ,20ꢀ,26-
pentahydroxy-10R,23R-cucurbit-5,24-(E)-dien-11-one.
Compound 2 showed a quasimolecular ion at m/z 703.3664 [M
+ Na]+ in the HRESIMS, indicating a molecular formula of
C36H56O12. Comparison of its 1H and 13C NMR data with those of
1 revealed that compound 2 was a glycoside of 1. This observation
was confirmed by enzymatic hydrolysis of 2 with ꢀ-glucosidase to
to carbons bearing oxygen. Analyses of its 1H-1H COSY and
HSQC spectra led to the fragments C-10-C-1-C-2-C-3; C-6-C-
7-C-8; C-15-C-16-C-17; and C-22-C-23-C-24. Comparison
of the NMR data of 3 with those of (2ꢀ,9ꢀ,10R,16R,23S)-16,23-
epoxy-2,20,26-trihydroxy-9-methyl-19-norlanosta-5,24-(Z)-diene-
3,11-dione37 indicated that the structure of 3 was similar to that
compound. According to the number of unsaturation equivalents,
the 16,23-epoxy function was open, which was supported by the
differences of chemical shifts of surrounding carbons and also by
the HMBC spectrum, in which13C-1H long-range correlation
signals were not observed at H-16/C-23 and H-23/C-16. 13C-1H
long-range correlation signals were observed at H-23/C-25 and
H-26/C-24, indicating one double bond at C-24/C-25 and that one
of the terminal methyl groups was hydroxylated. The ROESY
spectrum of 3 revealed the relative configuration of the tetracyclic
skeleton. Furthermore, the NOE correlation between CH2-26 and
H-24 revealed the E configuration of the olefinic bond. Compound
3 was thus established to be 2ꢀ,16R,20,23,26-pentahydroxy-10R-
cucurbit-5,24-(E)-diene-3,11-dione.
The positive HRESIMS of 4 showed the quasimolecular ion
signal at m/z 759.3568 [M + Na]+, and in conjunction with the
13C NMR data, its molecular formula was determined to be
C38H56O14, indicating 11 degrees of unsaturation. The 38 resonances
in its 13C NMR spectrum were consistent with a triterpenoid
backbone bearing a sugar moiety. Enzymatic hydrolysis of 4 gave
glucose and the aglycone, which was identified as cucurbitacin A
by comparison with spectroscopic data in the literature.30 13C-1H
long-range correlation signals at H-1glc/C-2 and H-2/C-1glc in its
HMBC spectrum indicated linkage of the glucose moiety to C-2
of the aglycone. The sugar moiety was determined to be ꢀ-D-
glucopyranose by spectroscopic and chemical methods as for
afford compound 1 and glucose. The specific rotation value of the
24
obtained glucose, [R]D +51 (c 0.035, H2O), indicated its
D
configuration. A significant downfield shift of C-2 from δC 69.3 in
1 to δC 76.9 in the 13C NMR spectrum suggested that the glucose
was at C-2, which was confirmed by the 13C-1H long-range
correlations of the anomeric proton at δH 4.38 with C-2 (δC 76.9)
and also H-2 (δH 4.12) with C-1glc (δC 102.4) in the HMBC
spectrum. The sugar unit was deduced to be a ꢀ-glycoside from
the coupling constant of the anomeric proton (J ) 7.7 Hz).
Accordingly, the structure of 2 was determined to be 16R,23R-
epoxy-2ꢀ,3ꢀ,7ꢀ,20ꢀ,26-pentahydroxy-10R,23R-cucurbit-5,24-(E)-
dien-11-one 2-O-ꢀ-D-glucopyranoside.
The molecular formula of 3 was determined to be C30H46O7 by
HRESIMS (positive-ion mode) ([M + Na]+, m/z 541.3120),
indicating eight degrees of unsaturation. Its 13C NMR spectrum
showed 30 resonances, comprising seven methyl, six methylene
(one oxygenated), eight methine (two sp2 and three oxygenated
methines), and nine quaternary carbons (two carbonyl and two sp2
carbons, one oxygenated quaternary carbon). Its 1H NMR spectrum
revealed the existence of two olefinic protons (δH 5.80, 1H, d, 3.5
Hz and δH 5.50, 1H, d, 8.8 Hz), seven tertiary methyl groups (δH
0.85, 1.05, 1.26, 1.33, 1.38, 1.40, 1.72), and five protons bonded

Cucurbitane-Type Triterpenoids from Cucumis melo
Journal of Natural Products, 2009, Vol. 72, No. 5 827
24
compound 2 {[R]D +57 (c 0.29, H2O)}. Thus, compound 4 was
vealed that an R,ꢀ-unsaturated ketone in their side chains and a
25-acetoxy group were important structural requirements for
cucurbitacin cytotoxicity, which was in agreement with a previous
study.42 Saturation of the conjugated ∆23,24 olefinic bond eliminated
the cytotoxicity.
The effects of all isolated compounds on the Jak/Stat3 signaling
pathway were also evaluated, but only cucurbitacin B (10)
significantly suppressed phosphotyrosine stat3 levels (IC50, 3 µM).
None of the other compounds showed such activity at up to 10
µM.
cucurbitacin A 2-O-ꢀ-D-glucopyranoside.
Compound 5 possessed the elemental composition C30H44O8 as
determined by HRESIMS and NMR analyses. Its 1H and 13C NMR
data were similar to those of cucurbitacin A, except for the absence
of an acetoxy moiety along with downfield shifts of C-24 (+3.7
ppm), 26-Me (+2.6 ppm), and 27-Me (+2.8 ppm) and an upfield
shift of C-25 to δ 70.1 (-8.4 ppm). The structure of 5 was
confirmed by analyses of 1H-1H COSY, HSQC, HMBC, and
ROESY spectra and determined to be 25-deacetylcucurbitacin A.
Compound 6 had the molecular formula C30H46O8 as determined
from HRESIMS and NMR analyses, indicating one less unsaturation
Experimental Section
1
degree than that of 5. Its H and 13C NMR data were similar to
General Experimental Procedures. Optical rotations were measured
with a Perkin-Elmer 241MC polarimeter. IR spectra were recorded using
a Perkin-Elmer 577 spectrometer. LR-ESIMS were measured using a
Finnigan LCQ-DECA instrument, and HR-ESIMS data were obtained
on a Mariner spectrometer. NMR spectra were recorded on a Bruker
AM 400 with TMS as internal standard, and chemical shifts are
expressed in δ (ppm). Preparative HPLC was carried out using a Varian
SD-1 instrument, equipped with a Merck NW25 C18 column (10 µm,
20 mm × 250 mm) and Prostar 320 UV/vis detector, and the preparative
HPLC fractions were analyzed by analytic TLC. Column chromato-
graphic separations were carried out by using MCI gel CHP-20P
(75-150 µm; Mitsubishi Chemical Industry Co., Ltd.), silica gel H60
(300-400 mesh), zcx-II (200-300 mesh) (Qingdao Haiyang Chemical
Group Corporation, Qingdao, China), and Sephadex LH-20 (Pharmcia
Biotech AB, Uppsala, Sweden) as packing materials. HSGF254 silica
gel TLC plates (Yantai Chemical Industrial Institute, Yantai, China)
and RP-18 WF254 TLC plates (Merck) were used for analytical purposes.
ꢀ-Cellulase was manufactured by Lizhu Dongfeng BioTech Co. Ltd.,
Shanghai, People’s Republic of China.
those of 5 except for the absence of a disubstituted olefinic bond
and the emergence of two methylene signals. In contrast to 5, the
signal for C-22 showed a strong downfield shift to δ 215.4 (+13.1
ppm), whereas the two sp2 methines were missing and seven sp3
methylenes were observed. Analysis of 2D NMR experiments,
including 1H-1H COSY, HSQC, and HMBC spectra, revealed that
compound 6 was 23,24-dihydro-25-deacetylcucurbitacin A.
Compound 7 was obtained as white, amorphous powder with a
molecular formula of C32H46O9. Comparison of its 1H and 13C NMR
data with those of cucurbitacin B revealed their structural similarity,
and the emergence of a proton signal at δH 4.10 and methine carbon
at δ 66.2 with the loss of a methylene signal at δ 25.3 indicated
the replacement of a methylene by an oxygenated methine in 7.
In the 1H-1H COSY spectrum of 7, cross-peaks were found
between the proton signal at δH 4.10 and the olefinic signal at δH
5.96, which indicated the presence of an additional OH group at
C-7. Furthermore, the appearance of H-8 (δH 2.10) as a singlet
provided no coupling constant between H-7 and H-8, corresponding
to a dihedral angle of approximately 90°, and led to the conclusion
that the 7-OH was ꢀ-oriented. This was supported by the NOE
signal between CH3-30 and H-7. Thus, compound 7 was determined
to be 7ꢀ-hydroxycucurbitacin B.
Plant Material. The aerial parts of Cucumis melo were purchased
from the Yulin Medicinal Materials Market in Guangxi, China, in
October 2007, which were collected in Anhui Province, China, and
identified by Professor Jingui Shen of Shanghai Institute of Materia
Medica, Chinese Academy of Sciences. A voucher specimen (No.
SIMM071028) was deposited in the Herbarium of Shanghai Institute
of Materia Medica, Chinese Academy of Sciences.
Compound 8 had the molecular formula C32H48O9, indicating
Extraction and Isolation. Powdered air-dried stems of C. melo (5
kg) were percolated at room temperature with 95% EtOH (20 L × 3)
within 9 days. After evaporation of EtOH in Vacuo, the aqueous residue
(1.5 L) was extracted successively with petroleum ether, CHCl3, ethyl
acetate, and 1-butanol (1.5 L × 3 each), yielding petroleum ether (30.0
g), CHCl3 (200.0 g), ethyl acetate (15.0 g), and 1-butanol extracts (45.0
g), respectively. The CHCl3 extract (200.0 g) was chromatographed
using MCI gel vacuum liquid chromatography (VLC) (10 cm i.d. ×
25 cm) eluted with H2O, 10%, 20%, 40%, 60%, 70%, 80%, and 100%
EtOH (each 1000 mL) to yield fractions C1 (27.8 g), C2 (100 g), C3
(20 g), and C4 (3.3 g). Fraction C1 was separated by VLC on a silica
gel column using a petroleum-acetone gradient (10:1 to 0:1) to give
subfractions C1A and C1B. Fraction C1A (6.3 g) was subjected to
column chromatography (CC) over silica gel eluted with CHCl3-MeOH
(8:1) and preparative HPLC eluted with MeOH-H2O (10 mL/min, 10%
to 90% MeOH within 90 min) to afford arvenin I (5 g). Fraction C1B
(3 g) was separated by HPLC eluted with a MeOH-H2O gradient (10%
to 100% MeOH within 100 min) to yield arvenin II (500 mg). Fraction
C2 (100 g) was subjected to CC over silica gel eluted with
petroleum-acetone (10:1, 8:1, 6:1, 5:1, 3:1, 1:1, and 0:1, each 2 L) to
give four subfractions: C2A (6.5 g), C2B (3.0 g), C2C (2.4 g), and
C2D (3.5 g). Fraction C2A (6.5 g) was subjected to CC over silica gel
eluted with CHCl3-MeOH (50:1) to give cucurbitacin R (450 mg) and
isocucurbitacin R (400 mg). Fraction C2B (3.0 g) was subjected to
CC over silica gel eluted with CHCl3-MeOH (20:1) to give cucurbi-
tacin A (12) (300 mg), cucurbitacin G (35 mg), cucurbitacin H (68
mg), and hexanorcucurbitacin D (98 mg), which were further purified
by preparative HPLC eluted with a MeOH-H2O gradient (10% to 100%
MeOH within 50 min). Fraction C2C (2.4 g) was subjected to CC over
silica gel eluted with CHCl3-MeOH (50:1), then purified by PTLC
(eluted with CHCl3-MeOH, 10:1, 20:1, 20:1) and Sephadex LH-20
eluted with EtOH to afford 3 (6 mg), 7 (25 mg), and 8 (8 mg). Fraction
C2D (3.5 g) was separated by HPLC eluted with a MeOH-H2O
gradient (10% to 100% MeOH within 100 min) and PTLC (developed
with CHCl3-MeOH 10:1) to yield 5 (15 mg) and 6 (9 mg). Fraction
1
one less unsaturation degree than that of 7. Its H and 13C NMR
data were similar to those of 7 except for the absence of a
disubstituted olefinic bond and the emergence of two methylene
signals. In contrast to 7, the signal for C-22 showed a strong
downfield shift to δ 213.8 (-11.4 ppm), whereas the two sp2
methines were missing and five sp3 methylenes were observed. The
configuration of C-7 was deduced from the coupling constant
between H-7 and H-8. Compound 8 was finally identified to be
23,24-dihydro-7ꢀ-hydroxycucurbitacin B by further analyses of 2D
NMR spectra.
Compound 9 had the molecular formula C30H44O10. Enzymatic
hydrolysis of 9 gave the aglycone, which was identified as
hexanorcucurbitacin D by comparison with spectroscopic data in
the literature.33 The sugar moiety was determined to be ꢀ-D-glucose.
13C-1H long-range correlation signals at H-1glc/C-2 and H-2/C-
1glc in its HMBC spectrum indicated linkage of the glucopyranose
moiety to C-2 of the aglycone. On the basis of the above data,
compound 9 was elucidated as hexanorcucurbitacin D 2-O-ꢀ-D-
glucopyranoside.
All 21 cucurbitane-type triterpenoids were evaluated for cytotoxic
activity against human non-small-cell lung cancer A549/ATCC and
human hepatocellular BEL-7402 cells in Vitro. Cucurbitacin B (10)
inhibited the proliferation of A549/ATCC and BEL-7402 cells with
IC50 values of 0.01 ( 0.001 and 0.008 ( 0.001 µM, respectively,
while cucurbitacin A (10) inhibited the proliferation of A549/ATCC
and BEL-7402 cells with IC50 values of 0.4 ( 0.13 and 0.3 ( 0.11
µM, respectively. The new compound 7 showed weak cytotoxicity,
with IC50 values of 3.21 ( 0.85 and 7.59 ( 0.92 µM, against A549/
ATCC and BEL-7402 cell lines. The other compounds exhibited
no cytotoxicity at up to 10 µM. Preliminary analyses of the
structure-activity relationships of these natural triterpenoids re-

828 Journal of Natural Products, 2009, Vol. 72, No. 5
Chen et al.
C3 (2.0 g) was subjected to CC over silica gel eluted with
petroleum-acetone (10:1) to afford cucurbitacin B (10) (125 mg) and
dihydrocucurbitacin B (200 mg). Fraction C4 (3.3 g) was also separated
by silica gel CC eluted with petroleum-acetone (10:1) to afford
dihydroisocucurbitacin B (60 mg). The ethyl acetate fraction (15.0 g)
was separated by VLC over a silica gel column using petroleum-
acetone (10:1 to 0:1) as eluent to give fractions E1A (3.6 g) and E1B
(2.2 g). Fraction E1A was separated by preparative HPLC eluted with
a EtOH-H2O gradient (0% to 100% EtOH within 100 min), then
chromatographed on silica gel eluted with CHCl3-MeOH (20:1) to
give 1 (20 mg) and 4 (50 mg). The 1-butanol fraction was subjected to
CC over MCI gel (10 cm i.d. × 15 cm) with H2O, 20%, 40%, 60%,
80%, and 100% EtOH (each 1000 mL) as eluent to give fractions B1
(2.1 g), B2 (8.0 g), B3 (4.4 g), and B4 (7.1 g). Fraction B1 (2.1 g) was
separated by HPLC eluted with a MeOH-H2O gradient (10% to 100%
MeOH within 100 min), then purified by PTLC (eluted with
CHCl3-MeOH, 10:1) to yield 2 (16 mg). Fraction B3 (4.4 g) was
chromatographed on silica gel eluted with CHCl3-MeOH (50:1 to 3:1),
then purified by HPLC eluted with a MeOH-H2O gradient (10% to
100% MeOH within 50 min) to give 9 (12 mg). Fraction B4 (7.1 g)
was chromatographed on silica gel eluted with CHCl3-MeOH (15:1
to 9:1) to give 19-norlanosta-5,24-dien-11-one (36 mg).
eluted with EtOH to yield the glucose (1 mg, yield 62.8%), which was
compared with authentic sugar samples by co-TLC. Identification of
D-glucose in each aqueous layer was carried out by comparing the
specific rotation of the liberated glucose with that of authentic
D-glucose.43 Compounds 4 (30 mg) and 9 (6 mg) were hydrolyzed in
the same way as for 2, and the aglycones of 4 (16 mg) and 9 (4 mg)
in the organic layer were purified by CC over Si gel eluted with
CHCl3-MeOH (25:1).
Bioassay Method. Cytotoxicity of compounds against non-small-
cell lung cancer A549/ATCC cells and human hepatocellular BEL-
7402 cells was determined using the sulforhodamine B (SRB) assay.
Cells were plated in a 96-well plate 24 h before compound treatment
and continuously exposed to different concentrations of compounds
for another 72 h. After treatment, cells were fixed and stained with
SRB as described in Monks et al.44 Bound SRB was solubilized with
10 mM Tris, and absorbance was measured at 565 nm.
Western Blotting. After drug treatment, cells were washed twice
with ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
and 1.8 mM KH2PO4, pH 7.4) and lysed in SDS sample buffer. Cell
lysates, containing equal amounts of protein, were separated by SDS-
PAGE and transferred to polyvinylidine difluoride membranes. After
being blocked in 5% nonfat milk in TBST (Tris-buffered saline with
0.1% Tween 20, pH 7.6), membranes were incubated with the primary
phospho-Stat3 and ꢀ-tubulin antibodies at 4 °C overnight and then
exposed to appropriate secondary antibodies for 2 h at room temper-
ature. Immunoreactive proteins were visualized using the enhanced
chemiluminescence system from Pierce Chemical (Rockford, IL).
23
Compound 1: white, amorphous powder; [R]D +137 (c 0.15,
MeOH); IR (KBr) νmax 3415, 2956, 2927, 1685, 1456, 1385, 1207,
1
1047 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
(positive-ion mode) m/z 541.3 [M + Na ]+; HR-ESIMS m/z 541.3148
[M + Na]+ (calcd for C30H46O7Na, 541.3141).
23
Compound 2: white, amorphous powder; [R]D +163 (c 0.15,
Acknowledgment. The investigation was supported by the New
Drug Basic Research Program of the Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (SIMM0709JC-11).
MeOH); IR (KBr) νmax 3417, 2931, 1685, 1456, 1381, 1209, 1078,
1
1041 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
(positive-ion mode) m/z 703.3 [M + Na]+; HR-ESIMS m/z 703.3664
[M + Na]+ (calcd for C36H56O12Na, 703.3669). Glucose: [R]D +51
24
Supporting Information Available: Figures indicating the main
signals in the HMBC and COSY spectra of 1, 3, 5, and 9, the main
NOE correlation signals (T) in the ROESY spectra of 1, 3, 5, and 7,
and 1D and 2D NMR spectra of compounds 1-9 are available free of
charge via the Internet at http://pubs.acs.org.
(c 0.035, H2O).
23
Compound 3: white, amorphous powder; [R]D +86 (c 0.15,
CHCl3); IR (KBr) νmax 3423, 2970, 2928, 1714, 1689, 1464, 1377, 1022
cm-1 1H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
;
(positive-ion mode) m/z 541.3 [M + Na]+; HR-ESIMS m/z 541.3120
[M + Na]+ (calcd for C30H46O7Na, 541.3141).
23
Compound 4: white, amorphous powder; [R]D +28 (c 0.13,
References and Notes
MeOH); IR (KBr) νmax 3423, 2979, 1718, 1689, 1629, 1371, 1080,
1
622 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
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(positive-ion mode) m/z 759.3 [M + Na]+; HR-ESIMS: m/z 759.3548
[M + Na]+ (calcd for C38H56O14Na, 759.3568). Glucose: [R]D +57
24
(c 0.29, H2O).
23
Compound 5: white, amorphous powder; [R]D +45 (c 0.10,
CHCl3); IR (KBr) νmax 3428, 2975, 2929, 1714, 1689, 1629, 1382, 1091,
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8182.
1
754 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
(positive-ion mode) m/z 555.3 [M + Na]+; HR-ESIMS m/z 555.2927
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[M + Na]+ (calcd for C30H44O8Na, 555.2934).
23
Compound 6: white, amorphous powder; [R]D +64 (c 0.15,
CHCl3); IR (KBr) νmax 3435, 2974, 2929, 1699, 1367, 1209, 1054 cm-1
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1H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS (positive-ion
mode) m/z 557.4 [M + Na]+; HR-ESIMS m/z 557.3091 [M + Na]+
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23
Compound 7: white, amorphous powder; [R]D +65 (c 0.23,
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1256, 1126, 1022, 985 cm-1; H NMR and 13C NMR data, Tables 1
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23
Compound 8: white, amorphous powder; [R]D +29 (c 0.22,
CHCl3); IR (KBr) νmax 3448, 2923, 2852, 1716, 1464, 1369, 1255, 1022,
1
756 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
(positive-ion mode) m/z 599.3 [M + Na]+; HR-ESIMS m/z 599.3173
[M + Na]+ (calcd. for C32H48O9Na, 599.3196).
23
Compound 9: white, amorphous powder; [R]D +80 (c 0.17,
MeOH); IR (KBr) νmax 3425, 2920, 2852, 1695, 1464, 1375, 1080,
1
1030 cm-1; H NMR and 13C NMR data, Tables 1 and 2; LR-ESIMS
(positive-ion mode) m/z 587.3 [M + Na]+; HR-ESIMS m/z 587.2812
[M + Na]+ (calcd. for C30H44O10Na, 587.2832). Glucose: [R]D +70
24
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NP800692T