Antiproliferative and apoptotic sesquiterpene lactones from Carpesium faberi

Article abstract of DOI:10.1016/j.bmcl.2010.10.138

Four new (1-4) and 13 known (5-17) sesquiterpene lactones along with two known diterpenes (18, 19) were isolated from the whole plant of Carpesium faberi. The new structures were elucidated by means of spectroscopic techniques and some chemical transforma

Full text of DOI:10.1016/j.bmcl.2010.10.138

Bioorganic & Medicinal Chemistry Letters 21 (2011) 366–372
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiproliferative and apoptotic sesquiterpene lactones from Carpesium faberi
Xu-Wen Li a, Liang Weng b, Xue Gao c, Yun Zhao a, Fei Pang b, Jian-Hui Liu c, Hong-Feng Zhang b,
Jin-Feng Hu a,
⇑
a Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics, Ministry of Education & Shanghai Key Laboratory of Brain
Functional Genomics, East China Normal University, No. 3663 Zhongshan Road N, Shanghai 200062, PR China
b Institute of Biomedical Sciences, School of Life Science, East China Normal University, No. 3663 Zhongshan Road N, Shanghai 200062, PR China
c Research Center of Medical Chemistry & Chemical Biology, Chongqing Technology and Business University, No. 19 Xuefu Ave., Nan’an District, Chongqing 400067, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new (1–4) and 13 known (5–17) sesquiterpene lactones along with two known diterpenes (18, 19)
were isolated from the whole plant of Carpesium faberi. The new structures were elucidated by means of
Received 6 August 2010
Revised 20 October 2010
Accepted 30 October 2010
Available online 5 November 2010
spectroscopic techniques and some chemical transformations to be pseudoguaian-1
O-b- -glucopyranoside (1), 4b,10 -dihydroxy-5 (H)-1,11(13)-guaidien-8 ,12-olide (2), 4b,10b-dihy-
droxy-5 (H)-1, 11(13)-guaidien-8b,12-olide (3), and (4S)-acetyloxyl-11(13)-carabren-8b,12-olide (4).
All isolates were tested against MCF-7 human breast cancer cells using the MTT assay. Among them,
the sesquiterpene lactones (except tomentosin 17) possessing an -methylene- -lactone moiety were
found to have in vitro antiproliferative activities, with IC50 values of 3.0–38.8 g/mL. The effects of four
a(H)-8a,12-olide-4b-
D
a
a
a
a
Keywords:
Carpesium faberi
Compositae
Sesquiterpene lactones
Antiproliferative
a
c
l
selected sesquiterpene lactones (guaianolide 2, carabranolide 4, pseudoguaianolide 9, eudesmanolide 13)
on the cell cycle were examined using flow cytometry (FCM).
Ó 2010 Elsevier Ltd. All rights reserved.
The genus Carpesium (Compositae) consists of about 20 species
worldwide, and most of them are distributed in Eastern Asia.1,2 It
has been well documented that sesquiterpene lactones with
different carbon frameworks are characteristic for Carpesium.2,3
Carpesium faberi Winkl. has been used as a folk medicine in
southwestern China due to its hemostatic, vermifugal, anti-inflam-
matory, and detoxifying properties.1,4 However, this plant has not
yet been phytochemically and pharmacologically investigated. As a
part of our ongoing project towards the discovery of novel anti-
tumor agents from plants,5 four pseudoguaianolides (1, 9–11), five
guaianolides (2, 3, 7, 8, 12), three carabranolides (4–6), three
eudesmanolides (13–15), a germacranolide (16), and one xanthan-
olide (17) together with an acyclic (18) and a labdane-type (19)
diterpene (Fig. 1), were obtained from the whole plant of
C. faberi.6,7 Comparing their MS, NMR data, and physical properties
with those of literature, the known compounds were identified as
mination of the new compounds, as well as their antiproliferative
and apoptotic activities against MCF-7 human breast cancer cells.
The molecular formula of compound 116 was determined to be
C
21H34O8 from the positive mode high resolution electrospray ion-
ization mass spectra (HR-ESIMS), which gave an [M+Na]+ ion peak
at m/z 437.2147 (calcd 437.2146). Its IR spectrum indicated the
presence of hydroxyl (3405 cmꢀ1) and
c )
-lactone (1771 cmꢀ1
groups. In the highfield 1H NMR spectrum of 1, one methyl group
singlet at d 0.94 (3H, s) and two methyl group doublets at d 1.16
(3H, d, J = 7.8 Hz) and 0.95 (3H, d, J = 6.4 Hz) were observed
(Table 1). The 13C and DEPT NMR spectra of 1 showed 15 carbon
signals classified as three methyls, four methylenes, six methines
(two oxygenated at d 93.3 and 84.2), and two quaternary carbons
(one carbonyl at d 182.9), in addition to six typical oxygen-bearing
carbons attributed to a glucopyranosyl unit [d 106.1 (CH, C-10), 75.3
(CH, C-20), 78.2 (CH, C-30), 71.6 (CH, C-40), 77.8 (CH, C-50), 62.8 (CH2,
C-60)] (Table 1). These data suggested that 1 should be a sesquiter-
pene glycoside with an aglycone structurally related to the known
pseudoguaianolide 9.2a,11
carabrol (5),3g,8 carabrone (6),3h,9 4b,10b-dihydroxy-5
a
(H)-1,11(13)-
guaidien-8
a
,12-olide (7),3c
4a,10b-dihydroxy-5a(H)-1,11(13)-
guaidien-8b,12-olide (8),10 2-desoxy-4-epi-pulchellin (9),2a,11 car-
abrolactone B (10),2a 2,3-dihydroaromaticin (11),12 4-epi-isoinuvi-
The planar structure of 1 was determined by detailed 1D and 2D
NMR (COSY, HSQC, and HMBC) spectroscopic analysis. In the COSY
NMR experiment (Fig. 2) of 1, a spin system [–CH(O)CH2CH2-
CHCH(CH3)CH2CH(O)CH(CHCH3)CH2–] in the aglycone was found
between H-4 at d 3.69 and H2-3 at d 1.99/1.58, between H2-3 and
H2-2 at d 1.72/1.45, between H2-2 and H-1 at d 1.63, between
H-1 and H-10 at d 1.74, between H-10 and Me-15 at d 0.95, be-
tween H-10 and H2-9 at d 2.27/1.25, between H2-9 and H-8 at d
4.47, between H-8 and H-7 at d 2.48, between H-7 and H-11 at d
scolide (12),8 telekin (13),3f ivalin (14),13
2a
,5a
-dihydroxy-11
a(H)-
eudesma-4(15)-en-8b,12-olide (15),3d carabrolactone
A
(16),2a
tomentosin (17),8,9 trans-phytol (18),14 and 8
a,15-dihydroxy-13E-
labdene (19).15 We herein report the isolation and structure deter-
⇑
Corresponding author. Tel.: +86 21 62237510; fax: +86 21 62606791.
E-mail addresses: hfzhang@bio.ecnu.edu.cn (H.-F. Zhang), jfhu@brain.ecnu.
edu.cn (J.-F. Hu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.138

X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
367
R2
15
10
R1
OH
14
1
H
10
1
10
1
5
A
3
B
3
O
3
O
O
5
5
7
C
7
12
7
12
12
11
11
11
13
O
H
R1
O
H
RO
14
O
H
OH
R2
15
13
13
2 R1 = CH3, R2 = OH
3 R1 = CH3, R2 = OH
8 R1 = OH, R2 = CH3
1
R = β-D-Glucoside
7 R1 = OH, R2 = CH3
1a R = H
H
14
H
R1
R2
O
2
8
7
10
5
12
O
4
O
O
1
S
11
3
15
R1
O
R2
H
O
H
R3
OH
13
4 R1 = OAc, R2 = H
5 R1 = OH, R2 = H
6 R1 = R2 = O
9
R1 = OH, R2 = R3 = H
10 R1 = R3 = OH, R2 = H
11 R1 = R2 = O, R3 = H
12
H
O
O
R1
O
O
O
H
O
O
HO
H
O
O
R2
R3
16
17
13 R1 = H, R2 = OH, R3 = exocyclic CH2
14 R1 = OH, R2 = H, R3 = exocyclic CH2
OH
15 R1 = OH, R2 = OH, R3
= β-CH3
OH
OH
H
19
18
Figure 1. Structures of compounds 1–19.
2.62, between H-11 and Me-13 at d 1.16, as well as between H-7
and H2-6 at d 1.94/1.51. The relative stereochemistry of the chiral
centers in the aglycone was deduced by extensive analysis of the
coupling patterns of the protons bound to the seven-membered
ring and by the NOE correlations in the NOESY NMR experiment.
Clear NOE correlations (Fig. 2) were observed between H-1 at d
1.63 (br dd, J = 11.1, 10.2 Hz) and H-9ax at d 1.24 (br dd, J = 12.2,
12.0 Hz), between H-1 and H-4, between H-1 and H-7, between
H-1 and Me-15, between H-9ax and H-7, between H-9ax and
Me-15, between H-6ax at d 1.51 (dd, J = 14.8, 12.7 Hz) and H-8 at
d 4.47, between H-6ax and Me-13, between H-6ax and Me-14, be-
tween H-7 and H-6eq at d 1.94 (1H, dd, J = 14.8, 5.6 Hz), between
H-8 and Me-14, between H-8 and H-9eq at d 2.27 (ddd, J = 13.2,
3.6, 3.6 Hz), as well as between H-9eq and H-10 at d 1.74. These
½
a 2D2
ꢁ
+52 (c 0.09, H2O)].17 Thus, compound 1 was elucidated to be
pseudoguaian-1 (H)-8 ,12-olide-4b-O-b- -glucopyranoside.
a
a
D
Based on their HR-ESIMS, the molecular formula C15H20O4 of
both compounds 219 and 320 were determined to be the same as
those of the known guaianolides 73c and 8.10 Compounds 2, 3, 7,
and 8, each possessing an
a-methylene-c-lactone moiety, were
found to have similar NMR spectral features (Table 2). The proton
and carbon signals of the diastereoisomers 2 and 3 were unambig-
uously assigned by 2D NMR experiments (COSY, HSQC, and HMBC).
Similar to 1, the relative stereochemistry of 2 and 3 was deduced
according to the coupling patterns of the protons bound to the se-
ven-membered ring and the NOE correlations in their NOESY NMR
experiments (Fig. 3). In the NOESY spectrum of 2, clear NOE corre-
lations were observed between H-7 at d 3.07 and H-5 at d 2.82, be-
tween H-7 and H-6eq at d 2.18 (ddd, J = 12.6, 4.6, 3.2 Hz), between
H-7 and H-9ax at d 2.08 (dd, J = 13.8, 11.2 Hz), between H-8 at
d 3.83 and H-6ax at d 1.01 (dd, J = 12.1, 12.1 Hz), between H-8
and H-9eq at d 2.49 (dd, J = 13.8, 4.4 Hz), between H-8 and Me-14
at d 1.58, between H-9eq and Me-14, between H-5 and Me-15 at
d 1.39, as well as between H-6eq and Me-15. These data showed
data revealed that H-1, H-4, H-7, H-11, and Me-15 were all a-ori-
ented, whereas H-8, Me-13, and Me-14 were in the b-orientation
(Figs. 1 and 2). Therefore, the A/B/C rings in the aglycone of 1 were
in turn trans-fused.
The glycosidic linkage position at C-4 was determined by the
HMBC NMR experiment (Fig. 2). A clear 3J correlation were found
between the anomeric proton at d 4.30 (1H, d) and C-4 resonating
at d 93.3. The b-glycosidic linkage was deduced based on the ob-
served coupling constant (7.8 Hz) of the anomeric proton. In addi-
tion, the enzymatic hydrolysis17 of 1 with b-glucosidase gave the
aglycone 1a18 (Table 1) and glucose. The relative stereochemistry
of 1a was further analyzed by its own NOESY NMR experiment,
that the c-lactone ring is trans-fused at C-7 and C-8 in compound
2. Therefore, 2 is the C-10 epimer of compound 7. Similarly
(Fig. 3), compounds 3 and 8 are epimers at C-4, and both com-
pounds have a cis-fused
compounds 2 and 3 were elucidated to be 4b,10
(H)-1,11(13)-guaidien-8 ,12-olide and 4b,10b-dihydroxy-5a
c
-lactone ring at C-7 and C-8. Therefore,
-dihydroxy-
(H)-
a
5
a
a
which was in full agreement with those of 1. The
of the glucose was identified by direct comparison with an authen-
tic sample using HPLC analysis and optical rotation [purified sugar:
D
-configuration
1,11(13)-guaidien-8b,12-olide, respectively. Interestingly, the
structure of 3 was once presented as a known compound by Hocine
Dendougui et al.;21 however, the structure (without any NMR data)

368
X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
Table 1
1H (500 MHz) and 13C (125 MHz) NMR Data of 1, 1a, and 4a
Position
1b
1ac
4c
dH (mult, J, Hz)
dC (DEPT)
dH (mult, J, Hz)
dC (DEPT)
dH (mult, J, Hz)
dC (DEPT)
1
2
1.63 brdd (11.1, 10.2)
1.72 m, overlapped
1.45 m
48.1 (CH)
26.7 (CH2)
1.58 brdd (10.3, 9.8)
1.73 m
1.43 m, overlapped
1.97 m
47.6 (CH)
25.3 (CH2)
0.43 m
1.30 m (2H)
34.7 (CH)
24.9 (CH2)
3
1.99 m
28.8 (CH2)
28.9 (CH2)
1.68 m
35.9 (CH2)
1.58 m
1.34 m
1.56 m
4
5
6
3.69 dd (9.2, 8.9)
93.3 (CH)
45.8 (C)
35.7 (CH2)
3.74 dd (9.4, 8.3)
82.2 (CH)
44.4 (C)
34.7 (CH2)
4.88 m
0.34 m
2.35 dd (14.1, 6.9, Heq
0.90 m
3.15 dddd (11.8, 9.2, 3.1, 3.0)
4.76 ddd (11.2, 9.2, 4.4)
2.31 dd (13.8, 4.4, Heq
70.7 (CH)
22.8 (CH)
30.8 (CH2)
1.94 dd (14.8, 5.6, Heq
1.51 dd (14.8, 12.7, Hax
2.48 m
4.47 ddd (11.2, 10.5, 3.3)
2.27 ddd (13.2, 3.6, 3.6, Heq
)
1.83 dd (14.6, 5.7, Heq
1.41 m, overlapped
2.41 m
4.36 ddd (11.3, 10.3, 3.4)
2.33 ddd (13.0, 3.7, 3.6, Heq
)
)
)
7
8
9
44.2 (CH)
84.2 (CH)
46.1 (CH2)
43.2 (CH)
83.4 (CH)
44.9 (CH2)
37.8 (CH)
75.6 (CH)
37.3 (CH2)
)
)
)
1.24 br dd (12.2, 12.0, Hax
1.74 m, overlapped
2.62 dq (7.9, 7.8)
)
1.23 br dd (12.2, 12.0, Hax
1.69 m
2.62 dq (7.9, 7.8)
)
0.94 dd (13.8, 11.2, Hax)
10
11
12
13
14
15
OAc
30.6 (CH)
41.4 (CH)
182.9 (C)
11.8 (CH3)
18.5 (CH3)
20.9 (CH3)
29.7 (CH)
39.8 (CH)
179.9 (C)
11.6 (CH3)
17.3 (CH3)
20.5 (CH3)
17.0 (C)
139.0 (C)
170.5 (C)
122.5 (CH2)
18.2 (CH3)
21.3 (CH3)
19.9 (CH3)
170.7 (C)
1.16 d (7.8)
0.94 s
0.95 d (6.4)
1.17 d (7.8)
0.87 s
0.93 d (6.5)
6.22 d (2.8), 5.54 d (2.3)
1.05 s
1.20 d (6.3)
2.01 s
10
20
30
40
50
60
4.30 d (7.8)
106.1 (CH)
75.3 (CH)
78.2 (CH)
71.6 (CH)
77.8 (CH)
62.8 (CH2)
3.19 dd (8.8, 7.8)
3.34 dd (9.0, 8.8)
3.29 dd (9.3, 9.0)
3.24 ddd (9.3, 5.6, 2.1)
3.83 dd (11.8, 2.1)
3.66 dd (11.8, 5.6)
a
b
c
Assignments were made by a combination of 1D and 2D NMR techniques (COSY, HSQC, and HMBC).
Recorded in CD3OD.
Recorded in CDCl3.
H
0.95
2.27
1.74
4.47
O
0.94
CH2OH
1.24
1.16
1.51
O
O
O
1.63
OH
1.94
2.48
3.69
OH
2.62
COSY
NOESY
OH
HMBC
Figure 2. Observed key COSY, HMBC, and NOE correlations of compound 1.
cited therein was not identical to the original one.10,21 Thus, 3 is
still a new naturally occurring compound.
acid (MTPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCꢂHCl) and 4-dimethylaminopyri-
dine (4-DMAP) to give its 4-(S)-MTPA ester (4b)26 and 4-(R)-
MTPA ester (4c),27 respectively. In their 1H NMR spectra, the
chemical shifts of Me-15 and H-4 in 4b could be observed at
The molecular formula C17H24O4 of compound 422 was deduced
from its positive mode HR-ESIMS, which resulted in an [M+Na]+ ion
peak at m/z 315.1587 (calcd 315.1567). Its 1H and 13C NMR data
(Table 1) showed that 4 is an analog of 5,3g,8 with an acetoxyl group
[dH: 2.01 (3H, s, H-17), dC: 19.9 (CH3), 170.7 (C@O)] in place of the
hydroxyl group at C-4 in the side chain of 5. Alcoholysis23 of 4 with
MeOH/MeONa gave 4a, which was found to be identical to
compound 5 by comparing their spectroscopic data and physical
properties. To the best of our knowledge, the stereochemistry of
C-4 in 4a (5) has not been previously assigned. Compound 4a
was then subjected to a modified Mosher’s method24,25 by treat-
higher fields (
tons resonated at a lower field (
D
dH = dS ꢀ dR: negative value), while the other pro-
D
dH: positive value) (Fig. 4) when
compared with those of 4c. This indicated an S configuration at
C-4 in 4a. Thus, compound 4 was elucidated as (4S)-acetyloxyl-
11(13)-carabren-12,8b-olide. Consequently, the structure of
5
was updated as (4S)-acetyloxyl-11(13)-carabren-8b,12-olide.
Compound 4 was detected in the MeOH extract of the title plant
by TLC and HPLC analysis, which indicated that 4 is not an artifi-
cial product.
ment with (S)- and (R)-a-methoxy-a-trifluoromethylphenyl acetic

X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
369
Table 2
1H (500 MHz) and 13C (125 MHz) NMR data of 2, 3, and 7 (in CDCl3)a
Position
2
3
7b
dH (mult, J, Hz)
dC (DEPT)
dH (mult, J, Hz)
dC (DEPT)
dH (mult, J, Hz)
1
2
3
151.8 (C)
125.0 (CH)
45.9 (CH2)
151.8 (C)
122.0 (CH2)
46.7 (CH2)
5.68 br s
2.51 d (17.4)
2.33 dd (17.4, 3.2)
5.57 br s
2.40 br s (2H)
5.88 dd (3.0, 1.6)
2.51 d (17.2)
2.31 dd (17.2, 3.2)
4
5
6
81.5 (C)
54.7 (CH)
32.0 (CH2)
81.3 (C)
55.4 (CH)
32.5 (CH2)
2.82 dd (12.6, 4.8)
2.18 ddd (12.6, 4.6, 3.2, Heq
1.01 br dd (12.2, 12.2, Hax
3.07 dddd (11.8, 9.2, 3.1, 3.0)
3.83 ddd (11.2, 9.2, 4.4)
2.70 br d (12.0)
2.36 dd (12.6, 3.1)
2.22 ddd (12.6, 3.5, 3.5, Heq)
1.07 br dd (12.4, 12.2, Hax)
2.65 dddd (11.5, 9.8, 3.3, 3.0)
3.97 ddd (11.2, 9.8, 1.4)
)
2.06 ddd (13.2, 4.7, 4.4, Heq
1.34 br dd, overlapped
3.39 m
5.15 ddd (11.7, 4.5, 3.5)
2.29 dd (13.8, 4.0, Heq
)
)
7
8
9
45.6 (CH)
81.8 (CH)
46.5 (CH2)
42.2 (CH)
77.5 (CH)
42.5 (CH2)
2.49 dd (13.8, 4.4, Heq
)
)
2.50 dd (14.2, 1.3, Heq)
2.08 dd (13.8, 11.2, Hax
)
1.76 dd (13.8, 12.1, Hax
)
2.07 dd (14.2, 11.3, Hax)
10
11
12
13
70.2 (C)
69.6 (C)
139.8 (C)
170.0 (C)
120.1 (CH2)
140.4 (C)
170.1 (C)
121.6 (CH2)
6.21 d (3.0)
5.52 d (3.0)
1.58 s
6.26 d (2.8)
5.59 d (2.4)
1.54 s
6.17 d (3.4)
5.46 d (3.1)
1.45 s
14
15
30.7 (CH3)
23.3 (CH3)
29.9 (CH3)
24.5 (CH3)
1.39 s
1.36 s
1.39 s
a
Assignments were made by a combination of 1D and 2D NMR techniques (COSY, HSQC, and HMBC).
Data were recorded in CDCl3 for the first time.
b
1.76
2.29
2.49
5.68
5.57
1.58
2.08
2.51
3.83
1.01
1.54
2.06
5.15
2.33
1.34
1.36
3.07
2.82
2.70
2.18
1.39
3.39
3
2
Figure 3. Observed key NOE correlations of compounds 2 and 3.
Table 3
+ 0.02
+ 0.02
+ 0.05
Effects of 1–19 against MCF-7 cells (mean SD, n = 3)
OR
O
+ 0.11
+ 0.16
Compound
IC50
>50
(lg/mL)
+ 0.02
+ 0.01
S
- 0.01
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
+ 0.06
31.7 1.6
38.8 0.5
9.4 0.3
11.4 0.3
10.6 0.6
31.9 1.6
26.5 0.6
9.8 0.4
10.7 0.1
3.9 0.2
16.3 1.1
3.0 0.1
12.5 0.7
>50
>50
>50
>50
18.6 0.4
14.1 0.7
0.5 0.1
+ 0.07
+ 0.05
- 0.08
+ 0.03
+ 0.01
H
+ 0.07
+ 0.01
+ 0.01
4b: R = (S)-MTPA
4c: R = (R)-MTPA
Figure 4. Results with the modified Mosher’s method (
D
dH = dS ꢀ dR).
The genus Carpesium is a rich source of sesquiterpene lac-
tones.2,3 From a chemotaxonomical point of view the isolation of
different types of sesquiterpene lactones (1–17) from C. faberi
may be of interest. Sesquiterpene lactone glycosides (e.g., 1) have
seldom been found from this genus.3e All isolated compounds
(1–19) were evaluated for their antiproliferative activities
(Table 3) against MCF-7 human breast cancer cells using the MTT
assay.5,28,29 The isolated sesquiterpene lactones (except xanthano-
17
18
19
5-Fluorouracil
Daunorubicin
lide 17) with an
a-methylene-c-lactone moiety exhibited

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X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
Figure 5. Cell cycle effects of compounds 2, 4, 9, and 13 on MCF-7 cells.
Acknowledgments
We gratefully acknowledge Professor Bao-Kang Huang (The
Second Military Medical University of PR China) for the plant iden-
tification. The authors thank Professor Yi-Hua Yu from Shanghai
Key Laboratory of Functional Magnetic Resonance Imaging (East
China Normal University) for his assistance with NMR data acqui-
sition. This work was supported by an NSFC grant (90713040), two
STCSM grants (07DZ22006, 06DZ19002), and a grant from the State
Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica (SIMM), Chinese Academy of Sciences (CAS).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.138.
References and notes
Figure 6. Effects of compounds 2, 4, 9, and 13 on MCF-7 cells after 24 h exposure
(mean SD, n = 3). p <0.05; p <0.01.
*
**
1. Chinese Academy of Sciences. Flora Reipublicae Popularis Sinicae; Scientific
Press: Beijing, 1977; Vol. 75, p 293.
2. (a) Wang, F.; Yang, K.; Ren, F. C.; Liu, J. K. Fitoterapia 2009, 80, 21; (b) Kim, D. K.;
Baek, N. I.; Choi, S. U.; Lee, C. O.; Lee, K. R.; Zee, O. P. J. Nat. Prod. 1997, 60, 1199.
3. (a) Gao, X.; Lin, C. J.; Jia, Z. J. J. Nat. Prod. 2007, 70, 830; (b) Yang, C.; Zhu, Y.; Jia,
Z. J. Aust. J. Chem. 2003, 56, 621; (c) Kim, M. R.; Kim, C. S.; Hwang, K. H.; Park, I.
Y.; Hong, S. S.; Son, J. K.; Moon, D. C. J. Nat. Prod. 2002, 65, 583; (d) Yang, C.; Shi,
Y. P.; Jia, Z. J. Planta Med. 2002, 68, 626; (e) Lin, Y. L.; Ou, J. C.; Kuo, Y. H.; Lin, J.
K.; Lee, K. H. J. Nat. Prod. 1996, 59, 991; (f) Dong, Y. F.; Ding, Y. M. Acta Bot. Sin.
1988, 30, 71; (g) Maruyama, M.; Karube, A.; Sato, K. Phytochemistry 1983, 22,
2773; (h) Maruyama, M.; Omura, S. Phytochemistry 1977, 16, 782; (i)
Maruyama, M.; Shibata, F. Phytochemistry 1975, 14, 2247.
significant in vitro antiproliferative activities, and compounds 11
and 13 were the most potent with an IC50 value of 3.9 and
3.0 l
g/mL, respectively. Cell cycle analysis30 indicated that when
MCF-7 cells were treated with four selected sesquiterpene lactones
(guaianolide 2, carabranolide 4, pseudoguaianolide 9, eudesmano-
lide 13) at IC50 concentrations for 24 h, the percentage of cells in
the G2/M phase was significantly increased (p <0.01) (Figs. 5 and
6), suggesting that the cancer cells were killed mostly at G2/M
phase of the cell cycle. Moreover, when treating MCF-7 cells with
compounds 2 and 4 at IC50 concentrations for 24 h, obvious
sub-G1 peaks (apoptosis >5%) were observed (Figs. 5 and 6),
demonstrating that these two new compounds may have apoptosis
effects on MCF-7 cells.
4. Jiangsu New Medical College
A Dictionary of Traditional Chinese Medicine;
Shanghai Science and Technology Press: Shanghai, 1986. p 308.
5. (a) Li, X. W.; Guo, Z. T.; Zhao, Y.; Zhao, Z.; Hu, J.-F. Phytochemistry 2010, 71, 682;
(b) Wu, S. B.; Pang, F.; Wen, Y.; Zhang, H. F.; Zhao, Z.; Hu, J.-F. Planta Med. 2010,
76, 82; (c) Wu, S. B.; Ji, Y. P.; Zhu, J. J.; Zhao, Y.; Xia, G.; Hu, Y. H.; Hu, J.-F. Steroids
2009, 74, 761.
6. Plant material: The whole plant of C. faberi was collected from Changshou
County, Chongqing, PR China in August 2008. The plant was identified by

X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
371
Professor Bao-Kang Huang (Department of Pharmacognosy, The Second
Military Medical University, Shanghai, PR China). A voucher specimen (no.
090523) was deposited at the Herbarium of the Shanghai Key Laboratory of
Brain Functional Genomics, East China Normal University.
the purified sugar with those of an authentic sample confirmed that the sugar
unit in 1 was -glucose. The EtOAc layer was also filtered and concentrated
D
under reduced pressure to furnish the aglycone 1a (3.8 mg), which was
purified by preparative TLC (PE–AC 3:1, Rf = 0.6).
7. Extraction and Isolation: The air-dried and pulverized plant material (3.6 kg)
was extracted with MeOH (5 L ꢃ 6) at room temperature (rt). After removal of
the solvent under reduced pressure, the entire residue (260.0 g) was subjected
to column chromatography (CC) over silica gel (column: 10 ꢃ 80 cm) with a
petroleum ether (PE)–acetone (AC) gradient (15:1—AC neat, v/v) followed by
AC–MeOH (10:1—MeOH neat, v/v) to yield five fractions (Fr.1–Fr.5). Fr.1 (PE–
AC 15:1, 12.9 g) was applied to a silica gel column (4.0 ꢃ 25 cm) and eluted
with PE–EtOAc (12:1) to give five subfractions (Fr.1.1–Fr.1.5). Fr.1.4 (108.8 mg)
was purified by gel permeation chromatography on Sephadex LH-20
(2.5 ꢃ 90 cm) in CH2Cl2–MeOH (2:1) to afford compound 18 (57.8 mg). Fr.2
(PE–AC 3:1, 25.8 g) was chromatographed on an MCI gel column (5.5 ꢃ 35 cm)
using a stepwise gradient elution with MeOH–H2O (from 1:2 to MeOH neat, v/
v) to generate five subfractions (Fr.2.1–Fr.2.5). Fr.2.2 (12.5 g) was subjected to a
silica gel column (4.0 ꢃ 25 cm) eluted with PE–AC (5:1) and further purified by
semi-preparative HPLC with an isocratic elution of MeOH–H2O (68:32, v/v)
over 20 min (flow rate: 3 mL/min) to afford compounds 11 (10.2 mg,
tR = 9.2 min) and 13 (55.2 mg, tR = 14.8 min). Fr.2.3 (69.3 mg) was also
purified by semi-preparative HPLC with an isocratic elution of MeOH–H2O
(52:48, v/v) for 30 min (flow rate: 3 mL/min) to afford compounds 6 (31.0 mg,
tR = 21.5 min) and 17 (6.9 mg, tR = 29.1 min). Fr.2.4 (3.5 g) was subjected to CC
over RP-C18 silica gel (column: 3.0 ꢃ 25 cm) eluted with a MeOH–H2O gradient
(from 1:1 to MeOH neat, v/v) to furnish compounds 5 (67.3 mg), 9 (161.9 mg),
and 12 (96.0 mg). Fr.3 (PE–AC 1:1, 3.3 g) was applied to a silica gel column
(2.5 ꢃ 25 cm) using CH2Cl2–AC (15:1) to give three subfractions (Fr.3.1–Fr.3.3).
Compounds 4 (48.8 mg) and 19 (4.2 mg) were purified from Fr.3.1 (165.5 mg)
by semi-preparative HPLC with an isocratic elution of MeOH–H2O (68:32, v/v)
over 25 min (flow rate: 3 mL/min; 4: tR = 18.5 min, 19: tR = 23.8 min). Fr.4 (AC–
MeOH 10:1, 25.8 g) was subjected to an MCI gel column (5.5 ꢃ 35 cm) eluted
with MeOH–H2O (from 1:4 to MeOH neat, v/v) to afford six subfractions
(Fr.4.1–Fr.4.6). Fr.4.2 (1.2 g) was chromatographed on a silica gel column
(3.0 ꢃ 25 cm) with PE–AC (2:1) and purified by semi-preparative HPLC to
afford compounds 7 (5.0 mg), 15 (5.6 mg), and 16 (7.1 mg). The method was an
isocratic elution of MeOH–H2O (23:77, v/v) over 15 min, followed by a linear
gradient of MeOH–H2O (23:77 ? 30:70, v/v) for 20 min (flow rate: 3 mL/min;
7: tR = 18.4 min, 15: tR = 27.5 min, 16: tR = 15.8 min). Fr.4.3 (9.2 mg) was
purified by semi-preparative HPLC with an isocratic elution of MeOH–H2O
18. Pseudoguaian-1
a
(H)-4b-hydroxyl-8
a
,12-olide (1a): Colorless gum; ½a D22
ꢀ15 (c
ꢁ
0.06, CDCl3); HRESIMS m/z = 275.1625 [M+Na]+ (calcd for C15H24O3Na,
275.1618,
Table 1.
D
= ꢀ2.5 ppm); for 1H and 13C NMR spectroscopic data, see
19. 4b,10
a
-Dihydroxy-5
a
(H)-1,11(13)-guaidien-8
a
,12-olide (2): White amorphous
power; ½a 2D2
ꢁ
ꢀ19 (c 0.05, CDCl3); UV (MeOH) kmax (log e) 217 (3.34); IR m
KBr
max
3356 (br), 2958, 2924, 2856, 1766, 1648, 1457, 1374, 1263, 1165, 1061, 1002,
711, 623 cmꢀ1
;
HRESIMS m/z = 287.1243 [M+Na]+ (calcd for C15H20O4Na,
287.1254,
Table 2.
D
= 3.8 ppm); for 1H NMR and 13C NMR spectroscopic data, see
20. 4b,10b-Dihydroxy-5
a
(H)-1,11(13)-guaidien-8b,12-olide (3): Colorless gum; ½a D22
ꢁ
KBr
ꢀ25 (c 0.04, CDCl3); UV (MeOH) kmax (log
e) 218 (3.40); IR
m
3412 (br), 2956,
max
2924, 2856, 1758, 1656, 1459, 1376, 1268, 1175, 1125, 1025, 996 cmꢀ1
;
HRESIMS m/z = 287.1239 [M+Na]+ (calcd for C15H20O4Na, 287.1254,
D
= 5.1 ppm); for 1H NMR and 13C NMR spectroscopic data, see Table 2.
21. Dendougui, H.; Benayache, S.; Benayache, F.; Connoly, J. D. Fitoterapia 2000, 71,
373.
22. (4S)-Acetyloxyl-11(13)-carabren-8b,12-olide (4): Colorless gum; ½ ꢁ +72 (c
a 2D2
KBr
0.30, CDCl3); UV (MeOH) kmax (log
e
) 220 (2.65); IR
m
3442 (br), 2935, 2865,
max
1763, 1733, 1661, 1461, 1373, 1246, 1145, 1035, 996, 950, 814 cmꢀ1; HRESIMS
m/z = 315.1557 [M+Na]+ (calcd for C17H24O4Na, 315.1567,
D H
= 3.1 ppm); for 1
and 13C NMR spectroscopic data, see Table 1.
23. Alcoholysis of compound 4: A solution of 4 (20.8 mg) in 2 mL CH2Cl2–MeOH (1:
20) was first treated with 5.5 mg MeONa and was then dehydrated by
Molecular Sieve 4A. The mixture was stirred at room temperature (rt) for 12 h
and then worked up as usual to give an oily residue of 4a (12.9 mg), which was
purified by preparative TLC (PE–AC 3:1, Rf = 0.4).
24. (a) Zhang, Z.; Zhang, W.; Ji, Y. P.; Zhao, Y.; Wang, C. G.; Hu, J.-F. Phytochemistry
2010, 71, 693; (b) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092.
25. Preparation of 4-O-(S)-MTPA ester (4b) and 4-O-(R)-MTPA ester (4c) of 4a: A
solution of 4a (3.8 mg) in dehydrated CH2Cl2 (1.5 mL) was treated with (S)-
MTPA (44.7 mg) in the presence of EDCꢂHCl (37.9 mg) and 4-DMAP (24.7 mg).
The mixture was stirred (rt) for 4 h and then worked up as usual to give an oily
residue of 4b. Compound 4b (3.5 mg) was purified by prep. TLC (PE–AC 5:1,
Rf = 0.4). Similarly, 4c (2.5 mg) was subsequently generated from 4a (2.7 mg)
using (R)-MTPA (32.4 mg) in a CH2Cl2 (1.5 mL) solution containing EDCꢂHCl
(41.7 mg) and 4-DMAP (37.2 mg).
(21:79, v/v) over 2 min, followed by
a linear gradient of MeOH–H2O
(21:79 ? 25:75, v/v) for 30 min (flow rate: 3 mL/min) to afford compound 8
(3.5 mg, tR = 24.5 min). Fr.4.4 (2.3 g) was subjected to a silica gel column
(3.0 ꢃ 25 cm) eluted with a CH2Cl2–MeOH gradient (20:1–5:1, v/v), and further
purified by semi-preparative HPLC with an isocratic elution of MeOH–H2O
(50:50, v/v) over 20 min (flow rate: 3 mL/min) to furnish compounds 2 (2.9 mg,
26. (4S)-11(13)-Carabren-8b,12-olide-4-O-(S)-MTPA ester (4b): HRESIMS m/z =
489.1840 [M+Na]+ (calcd for C25H29O5F3Na, 489.1859,
D
= 4.0 ppm); 1H NMR
(CDCl3, 500 MHz)
d 7.52–7.54 (2H, m), 7.40–7.41 (3H, m), 6.25 (1H, d,
J = 2.8 Hz, H-13), 5.56 (1H, d, J = 2.3 Hz, H-130), 5.15 (1H, m, H-4), 4.78 (1H, m,
H-8), 3.54 (3H, s, OCH3), 3.15 (1H, m, H-7), 2.36 (1H, m, H-6), 2.32 (1H, m, H-9),
1.79 (1H, m, H-3), 1.67 (1H, m, H-30), 1.39 (1H, m, H-2), 1.30 (1H, m, H-20), 1.28
(3H, d, J = 6.3 Hz, Me-15), 1.04 (3H, s, Me-14), 0.95 (1H, m, H-90), 0.89 (1H, m,
H-60), 0.43 (1H, m, H-1), 0.33 (1H, m, H-5).
tR = 14.8 min) and
3 (2.3 mg, tR = 16.8 min). Compound 1 (15.6 mg) was
purified from Fr.4.5 (30.5 mg) by semi-preparative HPLC with an isocratic
elution of MeOH–H2O (53:47, v/v) over 25 min (flow rate: 3 mL/min; 1:
tR = 20.4 min). Fr.4.6 (120 mg) was applied to a silica gel column (2.0 ꢃ 25 cm),
and purified by semi-preparative HPLC to afford compounds 10 (3.3 mg) and
14 (3.6 mg). The method was an isocratic elution of MeOH–H2O (46:54, v/v)
over 50 min, followed by an isocratic elution of MeOH–H2O (95:5, v/v) for
10 min (flow rate: 3 mL/min; 10: tR = 30.3 min, 14: tR = 47.7 min).
8. Bohlmann, F.; Mahanta, P. K.; Jakupovic, J.; Rastogi, R. C.; Natu, A. A.
Phytochemistry 1978, 17, 1165.
27. (4S)-11(13)-Carabren-8b,12-olide-4-O-(R)-MTPA ester (4c): HRESIMS m/
z = 489.1858 [M+Na]+ (calcd for C25H29O5F3Na, 489.1859,
D
= 0.3 ppm); 1H
NMR (CDCl3, 500 MHz) d 7.53–7.54 (2H, m), 7.39–7.40 (3H, m), 6.24 (1H, d,
J = 2.8 Hz, H-13), 5.56 (1H, d, J = 2.2 Hz, H-130), 5.16 (1H, m, H-4), 4.76 (1H, m,
H-8), 3.54 (3H, s, OCH3), 3.14 (1H, m, H-7), 2.33 (1H, m, H-6), 2.30 (1H, m, H-9),
1.72 (1H, m, H-3), 1.62 (1H, m, H-30), 1.28 (1H, m, H-2), 1.14 (1H, m, H-20), 1.36
(3H, d, J = 6.3 Hz, Me-15), 0.99 (3H, s, Me-14), 0.93 (1H, m, H-90), 0.88 (1H, m,
H-60), 0.37 (1H, m, H-1), 0.26 (1H, m, H-5).
9. Fontana, G.; La Rocca, S.; Passannanti, S.; Paternostro, M. P. Nat. Prod. Res. 2007,
21, 824.
10. Ahmed, A. A.; Mahmoud, A. A.; Williams, H. J.; Scott, A. I.; Reibenspies, J. H.;
Mabry, T. J. J. Nat. Prod. 1993, 56, 1276.
11. Zdero, C.; Bohlmann, F. Phytochemistry 1989, 28, 1653.
12. (a) Merfort, I.; Wendisch, D. Phytochemistry 1993, 34, 1436; (b) Bohlmann, F.;
Mahanta, P. K. Phytochemistry 1979, 18, 887.
13. (a) Topçu, G.; Öksüz, S.; Shieh, H.-L.; Cordell, G. A.; Pezzuto, J. M.; Candan, B.-J.
Phytochemistry 1993, 33, 407; (b) Herz, W.; Högenauer, G. J. Org. Chem. 1962,
27, 905.
28. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
29. Antiproliferative assay: MCF-7 cells were obtained from the American Type
Culture Collection (ATCC). The inhibition of the cell proliferation was assessed
by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide
(MTT)-based colorimetric assay. Briefly, MCF-7 cells were maintained in
Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA)
medium containing heat-inactivated 10% (v/v) fetal bovine serum (FBS)
(Bio-west, FRANCE), 100 unit/mL penicillin G sodium salt and 100 unit/mL
streptomycin sulfate (Gibco, Grand Island, NY, USA), in a 37 °C incubator under
an atmosphere of 5% CO2. The cells in exponential growth were placed at a final
concentration of 4.0 ꢃ 103 cells/well in a 96-well plate. After 24 h, the cells
were treated with compounds at varying concentrations. In order to exclude
phototoxicity, the process was kept away from bright light and the cells were
incubated in a dark incubator. After 48 h, the cells were incubated in fresh cell
14. Brown, G. D. Phytochemistry 1994, 36, 1553.
15. Rojatkar, S. R.; Chiplunkar, Y. G.; Nagasampagi, B. A. Phytochemistry 1994, 37,
1213.
16. Pseudoguaian-1
a
(H)-8
a
,12-olide-4b-O-b-
D
-glucopyranoside (1): White amor-
phous power; ½a 2D2
ꢁ
ꢀ54 (c 0.16, MeOH); IR
m
3405 (br), 2967, 2933, 2877,
KBr
max
1771, 1454, 1380, 1218, 1076, 1042, 994 cmꢀ1
; HRESIMS m/z = 437.2147
[M+Na]+ (calcd for C21H34O8Na, 437.2146,
spectroscopic data, see Table 1.
D
= ꢀ0.3 ppm); for 1H and 13C NMR
culture medium and washed carefully. Then, 100
added. After incubating for an additional 4 h, the supernatant was discarded
and 150 L DMSO (Sigma, USA) was added. After 0.5 h, the optical density
lL MTT (1 mg/mL) was
17. Enzymatic hydrolysis of compound 1: A solution of 1 (8.0 mg) in 0.2 M acetic acid
and sodium acetate buffer (pH 5.0, 1.5 mL) was treated with lyophilized
almond b-glucosidase (28.2 mg), and was then stirred at 40 °C for 6 h. After
cooling, the reaction mixture was partitioned into EtOAc. The aqueous layer
was filtered and concentrated under reduced pressure. The residue was
purified by gel permeation chromatography on Sephadex LH-20 (2.5 ꢃ 90 cm)
in MeOH to afford a mono-sugar (2.1 mg), which was then analyzed by HPLC
[column: Waters Sugar-pak 1 (300 ꢃ 6.5 mm); column temperature: 70 °C;
detector: Sedex 80 (SEDERE, France) evaporative light-scattering detector
(ELSD); mobile phase: H2O; flow rate: 0.5 mL/min]. Comparison of the
retention time (tR) in the HPLC-ELSD chromatogram and the optical datum of
l
(O.D.) of each well was measured at the wavelength of 570 nm using a Power
Wave XS microplate reader (Bio-Tek, Germany). 5-Fluorouracil (Sigma, USA)
and daunorubicin (Pharmacia Italia S.p.A., Italy) were used as the positive
controls, whereas 0.5% DMSO was used as the negative control. The purity of
the positive controls and tested compounds ranging from 95.0% to 99.9% was
determined by analytical HPLC with ELSD detection. The IC50 (the drug
concentration reducing by 50% the absorbance in treated cells, with respect to
untreated cells) values were calculated from the curves generated by plotting
the percentage of the viable cells versus the test concentration on a logarithmic
scale using SigmaPlot 10.0 software.

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X.-W. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 366–372
30. Flow cytometric (FCM) analysis: MCF-7 cells were used to analyze the cell cycle
effects of compounds 2, 4, 9, and 13. MCF-7 cells were seeded at 10 ꢃ 104 cells
per well in six-well culture plates. After 24 h, the cells were treated with tested
compounds at concentrations equivalent to their IC50 values, and incubated for
then treated with RNAse (100 lg/mL) for 20 min, stained with propidium
iodide (Sigma, USA) for 10 min, and finally analyzed using a FACS Calibur flow
cytometer (Becton Dickson, USA). The percentages of cells in G1, S, and G2/M
phases were determined by ModFit software (Verity Software House). All
experiments were performed in triplicate and gave the similar results.
an additional 24 h. Control samples included 0.5% DMSO and 1.8
lg/mL
daunorubicin. The cells were then fixed with 70% ethanol at 4 °C overnight,