Eudesmane-type sesquiterpene derivatives from Laggera alata

Article abstract of DOI:10.1016/j.phytochem.2013.07.014

Ten eudesmane-type sesquiterpene derivatives (1-10), including six cuauhtemone derivatives (1-6), one di-norsesquiterpene (3-oxo-di-nor-eudesma-4- en-11-oic acid, 7), and three eudesmane glycosides (alatoside F-H, 8-10) were isolated from the whole plants

Full text of DOI:10.1016/j.phytochem.2013.07.014

Phytochemistry 96 (2013) 201–207
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Eudesmane-type sesquiterpene derivatives from Laggera alata
Guo-Cai Wang ,1, Guo-Qiang Li 1, Hua-Wei Geng, Tao Li, Jiao-Jiao Xu, Fang Ma,
⇑
⇑
Xia Wu, Wen-Cai Ye, Yao-Lan Li
Instituent of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, China
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Jinan University, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten eudesmane-type sesquiterpene derivatives (1–10), including six cuauhtemone derivatives (1–6), one
di-norsesquiterpene (3-oxo-di-nor-eudesma-4-en-11-oic acid, 7), and three eudesmane glycosides
(alatoside F–H, 8–10) were isolated from the whole plants of Laggera alata together with 12 known com-
pounds. Their structures were elucidated on the basis of extensive spectroscopic analysis, acid hydrolysis,
and compounds 1 and 7 were studied by single-crystal X-ray diffraction analysis. The absolute configu-
ration of 1 was determined by the application of the modified Mosher’s method. All of the isolated eudes-
mane-type sesquiterpenes were evaluated for their cytotoxic activities on six human cancer cell lines, but
Received 8 December 2012
Received in revised form 20 February 2013
Available online 15 August 2013
Keywords:
Laggera alata
Compositae
Eudesmane
Sesquiterpene
all of the compounds were inactive on the tested cell lines in the concentration of 100
lg/mL.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The genus Laggera (Compositae), consisting of about 20 species,
is mainly distributed in tropical Africa and Southeast Asia (Zheng
et al., 2003b). Laggera alata (D. Don) Sch. Bip. ex Oliv. and L. ptero-
donta (DC.) Benth are the only two species of Laggera found in Chi-
na, and both are employed as traditional herbal medicines due to
their anti-inflammatory and anti-bacterial activities (Deng, 1963).
Previous studies on the genus Laggera established that its plants
contained a variety of compounds, whose major constituents were
eudesmane-type sesquiterpenes (Ahmed and Mahmoud, 1998; Li
et al., 2007; Onayade et al., 1990; Raharivelomanana et al., 1998;
Zhao et al., 1997a, 1997b; Zheng et al., 2003a, 2003b). As part of
our work on searching for bioactive constituents from traditional
herbal medicines, 10 new (1–10) and 12 known (11–22) eudes-
mane-type sesquiterpene derivatives were isolated from the whole
plants of L. alata. In 1addition, the cytotoxic effects of all the iso-
lated compounds on a panel of human cancer cell lines were eval-
uated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay, but these were inactive. This paper describes
the isolation, structure elucidation, and cytotoxic investigation
results of the eudesmane-type sesquiterpene derivatives from L.
alata.
The 95% EtOH extract of the whole plants ofL. alata was subjected
to macrosporous resin, silica gel, and Sephadex LH-20 column chro-
matography (CC), as well as preparative purification procedures
HPLC to give compounds 1–22. The structures of the new com-
pounds 1–10 (Fig. 1) were elucidated by the extensive spectroscopic
analysis, acid hydrolysis, and single-crystal X-ray diffraction analy-
sis. In addition to ten new compounds, the twelve known ones were
identified as pterodonoic acid (11) (Cui et al., 1998), 15-hydroxyi-
socostic acid (12) (Dawidar and Metwally, 1985), b-isocostic acid
(13) (Bohlmann et al., 1977), 1b-hydroxy-eudesma-4,11(13)-dien-
12-oic acid (14) (Ohmoto et al., 1987), 5a-hydroxy-4a,15-dihydro-
costic acid (15) (Zdero et al., 1990), 2
a
-hydroxylilicic acid (16)
(Abu Zarga et al., 1998), 5b-hydroxylilicic acid (17) (Zheng et al.,
2003a), ilicic acid (18) (Herz et al., 1966), (4S⁄,5E,10R⁄)-7-oxo-tri-
nor-eudesm-5-en-4b-ol (19) (Cheng et al., 2009), costic acid (20)
(Herz et al., 1966), 5a-hydroxycostic acid (21) (Zdero et al., 1990),
and cuauhtemone (22) (Nakanishi et al., 1974), respectively, by
comparing their NMR and MS data with literature data.
The HRESIMS of 1 indicated a quasi-molecular ion peak at m/z
475.2310 [M+Na]+ corresponding to the molecular formula of
C
24H26O8 (calcd for C24H26O8Na, 475.2302). The 13C NMR spectrum
showed the presence of 24 carbon resonances, consistent with the
putative molecular formula, including an ,b-unsaturated ketone
a
⇑
Corresponding authors at: Instituent of Traditional Chinese Medicine and
Natural Products, Jinan University, Guangzhou 510632, China. Tel.: +86 20
85221728; fax: +86 20 85221559.
at dC 201.9, two olefinic carbons at dC 146.4, 130.4, and three ester
carbonyls at dC 170.2, 169.8, 168.9. Analysis of the 1H and 13C NMR
spectroscopic data of 1 (Tables 1 and 2) suggested that 1 was a
cuauhtemone derivative. Four singlet methyls at dH 1.85, 2.06,
0.96 and 1.26 were assigned to H-12, H-13, H-14 and H-15 of the
E-mail addresses: wang_guocai@hotmail.com (G.-C. Wang), tliyl@jnu.edu.cn
(Y.-L. Li).
1
These authors contributed equally to this work.
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.07.014

202
G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
14
1
9
6
12
O
1
9
6
4'
2
3
O
O
10
2
3
8
8
O
10
12
5
2'
7
3'
1'
7
R3O
O
OH
4
11
13
O
5
H
AcO
O
AcO
4
11
R2O
R1O
H
OH
15
5'
HO
O
13
1 R1 = H R2 = Ac R3 = Ac
6
7
2
R1 = Ac R2 = H R3 = Ac
3 R1 = Ac R2 = Ac R3 = Ac
4 R1 = H R2 = H R3 = Ac
5
R1 = Ac R2 = H R3 = H
R2
R1
R3
O
COOH
COOH
R2
R1
OR3
R1
R2
10
R1 = O-β-D-Glc R2 = CH3
15 R1 = H R2 = α-OH R3 = H
16 R1 = OH R2 = α-H R3 = OH
17 R1 = OH R2 = β-OH R3 = H
8 R1 = O R2 = H R3 = β-D-Glc
12 R1 = H R2 = CH2OH
13 R1 = H R2 = CH3
14
9
R1 = H R2 = O R3 = β-D-Glc
11 R1 = O R2 = H R3 = H
18
R1 = OH R2 = α-H R3 = H
R1 = OH R2 = CH3
O
O
COOH
OH
R
HO
H
HO
19
22
20 R = H
21 R = OH
Fig. 1. Chemical structures of compounds 1–22.
Table 1
1H NMR spectroscopic data of compounds 1–6 (J in Hz).
No.
1
1a
2a
3b
4b
5b
6b
1.32 m
1.48 m
1.79 m
1.81 m
5.01br s
1.91 dd (13.5, 4.0)
2.21 m
3.00 dd (15.4, 3.3)
2.21 m
1.19 m
1.31 m
1.81 m
1.90 m
5.83 br s
2.15 m
2.20 m
2.84 d (12.9)
2.18 m
1.80 s
1.26 m
1.39 m
1.71 m
2.08 m
5.81 br s
2.14 m
2.22 m
2.89d (14.8)
2.21 m
1.85 s
1.29 m
1.45 m
1.81 m
2.04 m
4.91 br s
1.93 m
2.18 m
2.94 dd (15.3, 3.8)
2.23 m
1.83 s
1.29 m
1.54 m
1.81 m
1.90 m
5.91 br s
2.25 m
2.20 m
2.89 d (12.0)
2.25 m
1.86 s
1.26 m
1.50 m
1.81 m
1.90 m
5.90 br s
3.00 d (1.5)
6.88 d (1.5)
2
3
5
6
9
12
2.34 m
1.45 s
1.85 s
13
2.06s
2.00 s
2.07 s
2.04 s
2.07 s
1.45 s
14
0.96 s
0.93 s
0.97 s
0.95 s
1.00 s
0.99 s
15
1.26 s
1.53 s
1.58 s
1.28 s
1.60 s
1.55 s
30
5.23 q (6.4)
1.25 d (6.4)
1.66 s
5.04 q (6.2)
1.25 d (6.2)
1.29 s
5.12 q (8.1)
1.17 d (8.1)
1.62 s
5.13 q (6.4)
1.31 d (6.4)
1.41 s
3.95 q (6.4)
1.25 d (6.4)
1.30 s
5.11 q (6.4)
1.30 d (6.4)
1.33 s
40
50
AcO-4
AcO-20
AcO-30
2.07 s
2.07 s
1.91 s
2.07 s
2.05 s
2.05 s
1.96 s
1.98 s
1.93 s
1.99 s
1.99 s
a
Measured at 500 MHz in CDCl3.
Measured at 400 MHz in CDCl3.
b
presumed cuauhtemone moiety on the basis of HMBC correlations,
respectively. The downfield shifted proton at dH 5.01 (H-3) indi-
cated that the hydroxyl group at C-3 was esterified, which was fur-
ther confirmed by the correlation between H-3 and C-10 (dC 168.9)
in the HMBC spectrum. In addition, the HMBC correlation from
H-30 (dH 5.23) to carbonyl group (dC 169.8), and the comparison
of downfield shifted signals at dH 5.23 (H-30) and dC 82.1 (C-20) with
the reported data (Ahmad et al., 1991) indicated the side-chain was
a 20,30-diacetoxy-20-methylbutyryloxy group. The methyl groups of
the ester side-chain showing a singlet at dH 1.66 and a characteris-
tic doublet at dH 1.25 (J = 6.4 Hz) were attributed to H-50 and H-40,
respectively. The NOESY correlations of Hb-1/H-14, Hb-1/H-3, H-3/
H-15 and H-14/H-15 suggested that H-3, H-14 and H-15 were on
the same side of the cuauhtemone skeleton. Similarly, the NOESY
correlation between H -1 and H-5 placed H-5 on the opposite side
a
of the cuauhtemone skeleton. The structure and relative configura-
tion of 1 were further confirmed by single crystal X-ray diffraction
analysis (Fig. 2). The absolute configuration of C-3 in 1 was

G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
203
Table 2
13C NMR spectroscopic data of compounds 1–10.
No.
1a
2a
3b
4b
5b
6b
7a
8c
9c
10c
1
2
3
4
5
6
7
8
33.8
24.0
78.4
72.1
47.6
25.5
130.4
201.9
60.1
35.9
146.4
23.1
23.7
18.7
17.2
168.9
82.1
72.8
14.6
20.8
32.3
22.9
74.5
82.9
45.0
25.7
130.0
201.7
59.7
35.8
145.5
22.7
23.5
18.8
17.8
173.7
75.7
74.2
13.1
22.1
33.0
22.4
74.2
82.7
45.2
25.8
130.0
201.3
60.4
35.7
146.3
23.0
23.6
19.1
17.9
168.0
82.2
72.4
14.4
16.1
33.3
23.9
79.0
72.3
46.9
25.5
130.5
201.9
59.9
36.0
145.5
22.8
23.6
18.6
21.5
174.8
76.2
74.3
13.3
22.3
33.0
22.8
71.7
83.1
45.3
25.8
130.2
201.5
60.2
35.9
145.7
22.8
23.6
19.0
18.0
175.0
77.2
74.5
16.7
22.0
31.7
23.0
73.9
81.5
48.6
145.4
140.7
200.0
57.7
39.0
71.7
29.1
28.7
18.1
18.9
173.6
75.8
74.3
13.2
22.1
37.2
33.7
198.9
129.9
159.1
29.4
43.1
24.3
40.8
35.6
179.6
22.3
11.0
38.4
34.5
201.5
129.9
164.5
34.6
41.5
28.2
43.0
37.0
145.4
166.8
125.9
22.7
11.0
96.1
74.0
78.9
71.1
78.2
62.3
36.2
28.2
33.0
145.8
126.7
32.7
41.5
37.0
218.3
48.4
134.9
167.0
125.3
22.5
19.1
96.0
74.0
78.9
71.1
78.2
62.3
79.0
27.9
33.0
134.6
125.5
28.7
41.4
32.7
40.1
40.6
146.3
167.2
125.0
17.9
19.2
95.9
74.0
78.8
71.0
78.1
62.3
9
10
11
12
13
14
15
10
20
30
40
50
60
AcO-4
169.4
22.1
169.4
23.6
170.1
21.1
169.8
21.1
169.5
22.1
169.2
22.1
AcO-20
AcO-30
170.2
21.1
169.8
21.1
170.0
20.9
169.8
21.0
170.0
21.0
a
b
c
Measured at 125 MHz in CDCl3.
Measured at 100 MHz in CDCl3.
Measured at 125 MHz in CD3OD.
Fig. 2. X-ray crystal structures of 1 and 7.
(Table 2) of the two compounds were similar, except that C-4 of
1 (dC 72.1) was shifted downfield to dC 82.9 in 2, and likewise,
C-20 of 1 (dC 82.1) was shifted upfield to dC 75.7 in 2. This suggested
that the two acetoxy groups in 2 were at the C-4 and C-30 positions.
Therefore, compound 2 was determined as 4-O-acetyl-3-O-(30-
acetoxy-20-hydroxy-20-methylbutyryl)-cuauhtemone.
0.120
0.030
0.017 0.094
O
0.043
RO
-0.027
H
RO
-0.017
-0.060
The molecular formula of compound 3 was established as
22a R = (R)-MTPA
22b R = (S)-MTPA
C
26H38O9 by a quasi-molecular ion at m/z 517.2406 [M+Na]+ (calcd
for C26H38O9Na, 517.2408) in HRESIMS. The NMR spectroscopic
data (Tables 1 and 2) of 3 were very similar to those of 1, except
that 3 had the signals of an additional acetyl group (dC 170.1,
21.0), and the carbon resonance at C-4 shifted downfield from dC
72.1 in 1 to dC 82.7 in 3, suggesting the hydroxyl group at C-4
was esterified. The configuration of 3 was determined as same as
that of 1 by the coupling constants and NOESY correlations. So
the structure of 3 was elucidated as 4-O-acetyl-3-O-(20,30-diacet-
oxy-20-hydroxy-20-methylbutyryl)-cuauhtemone.
Fig. 3.
Dd Values (dS–dR) of the MTPA Esters 22a and 22b.
determined as R by acid hydrolysis and the modified Mosher’s
method (Fig. 3) (Su et al., 2002). Accordingly, compound 1 was de-
fined as 3-O-(20,30-diacetoxy-20-methylbutyryl)-cuauhtemone.
Compound 2 was isolated as a brown oil. The HRESIMS indi-
cated that 2 was an isomer of 1. The 13C NMR spectroscopic data

204
G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
Table 3
1H NMR spectroscopic data of compounds 7–10 (J in Hz).
No.
1
7a
8b
9b
10b
1.78 m
1.81 m
1.47 td (13.1, 4.3)
1.81 dt (13.1, 3.0)
1.73 m
2.38 m
1.68 td (13.1, 3.0)
3.40 m
2
3
6
2.42 m
2.53 m
2.35 dt (16.7, 3.8)
2.57 m
1.68 m
2.15 m
1.98 m
1.57 m
1.68 m
2.35 tt (12.3, 3.1)
1.80 t (13.2)
2.67 dt (13.2, 2.5)
1.20 td (13.2, 4.5)
2.06 dt (13.2, 3.1)
2.29 d (15.0)
2.99 d (15.0)
2.46 m
1.87 m
1.98 m
1.44 td (13.6, 3.9)
1.72 dt (13.6, 3.2)
1.24 s
2.13 m
2.93 m
2.61 m
1.81 m
1.74 m
1.75 m
1.51 m
1.95 m
2.72 d (14.3)
2.41 m
2.43 m
2.65 m
7
8
9
12
13
1.79 s
6.39 s
5.78 s
6.32 s
5.84 s
6.34 s
5.74 s
14
15
10
20
30
4
1.28 s
1.75 s
5.58 d (7.8)
3.40 m
3.42 m
3.38 m
3.41 m
3.84 dd (12.2, 1.6)
3.68 dd (12.2, 4.6)
1.26 s
1.73 s
5.57 d (7.9)
3.39 m
3.38 m
3.37 m
3.43 m
3.84 dd (12.2, 1.7)
3.68 dd (12.2, 4.7)
1.02 s
1.59 s
5.57 d (7.9)
3.39 m
3.41 m
3.38 m
3.41 m
3.84 dd (12.2, 1.8)
3.69 dd (12.2, 4.8)
50
60
a
Measured at 500 MHz in CDCl3.
Measured at 500 MHz in CD3OD.
b
The HRESIMS of compounds 4 and 5 showed that they had a
an allylic acid moiety in 11. This conclusion was confirmed by a
single crystal X-ray diffraction analysis (Fig. 2). Thus, compound
7 was determined as 3-oxo-di-nor-eudesma-4-en-11-oic acid.
The molecular formula C21H30O8 of compound 8 was deduced
from HRESIMS which showed a quasi-molecular ion peak at m/z
433.1831 [M+Na]+ (calcd for C21H30O8Na, 433.1833). Comparison
of the NMR spectroscopic data (Tables 2 and 3) of 8 with those
of pteodonoic acid (11) (Cui et al., 1998) showed that they were
very similar except for the presence of an additional glucose moi-
ety (dC 96.1, 74.0, 78.9, 71.1, 78.2, 62.3) in 8. The HMBC correlation
from H-10 (dH 5.58) to C-12 (dC 166.8) suggested that the sugar
same molecular formula of C22H34O7 by quasi-molecular ions at
m/z 433.2191 and 433.2195 [M+Na]+ (calcd for C22H34O7Na,
433.2197). The NMR spectroscopic data of 4 and 5 (Tables 1 and
2) were also similar to those of 2, except for the presence of only
one acetyl group in 4 and 5. Comparison of the chemical shifts at
C-4 and C-30 in 4 (dC 72.3, 74.3) and 5 (dC 83.1, 71.7) with those
in 2 (dC 82.9, 74.2) suggested that the acetyl group was connected
to C-30 in 4 and C-4 in 5, respectively. Thus, compounds 4 and 5
were determined as 3-O-(30-acetoxy-20-hydroxy-20-methylbuty-
ryl)-cuauhtemone and 4-O-acetyl-3-O-(20,30-dihydroxy-20-methyl-
butyryl)-cuauhtemone, respectively.
moiety was connected to C-12. Acid hydrolysis of 8 yielded D-glu-
The NMR spectroscopic data of compound 6 (Tables 1 and 2)
were similar to those of 2. The 1H NMR spectrum showed the pres-
ence of one olefinic proton at dH 6.88 (H-6), and four methyl groups
at dH 1.45 (H-12), 1.45 (H-13), 0.99 (H-14), and 1.55 (H-15) attrib-
utable to an eudesmane skeleton. The 13C NMR and DEPT spectra
displayed signals for two olefinic carbons at dC 145.4 and 140.7,
and three oxymethines at dC 71.7, 73.9, and 81.5. Additionally, a
methylene at dC 25.7 (C-6) in a cuauhtemone skeleton was missing.
These data suggested that 6 had a skeleton of 11-hydroxy-6,7-
dehydroeudesman-8-one (Vera et al., 2008). The NMR signals of
the remaining ester side-chains resembled those in 2, suggesting
the locations and compositions of the side-chains. These were fur-
ther confirmed by the HMBC correlations from H-3 (dH 5.90) to C-10
(dC 173.6), and from H-30 (dH 5.11) to the carbonyl group (dC 170.0).
cose which was identified by reversed-phase HPLC after conversion
of the sugar to thiocarbamoyl-thiazolidine derivative (Tanaka et al.,
2007). The large J value (7.8 Hz) of the anomeric proton at dH 5.58
indicated the glucopyranosyl moiety was b configuration. Thus,
compound 8 was determined to be b-
onoate, and named alatoside F.
D-glucopyranosyloxyl pteod-
The HRESIMS of compound 9 exhibited a quasi-molecular ion
peak at m/z 433.1835 [M+Na]+ (calcd for C21H30O8Na, 433.1833),
indicating the molecular formula was C21H30O8. The 1H and 13C
NMR spectroscopic data (Tables 2 and 3) were similar to those of
9-oxo-4,11(13)-eudesmadien-12-oic acid (Guerreiro et al., 1990;
Rustaiyan et al., 1987), except for the presence of an additional
glucose moiety (dC 96.0, 74.0, 78.9, 71.1, 78.2, 62.3). The HMBC cor-
relation from H-10 (dH 5.57) to C-12 (dC 167.0) confirmed that the
sugar moiety was connected to C-12. Acid hydrolysis of 9 yielded
Therefore,
6 was elucidated as 4a-acetoxy-3a
-(30-acetoxy-20-
methylbutyryloxy)-11-hydroxy-6,7-dehydroeudesman-8-one.
Compound 7 was isolated as a colorless block crystal. Its molec-
ular formula was determined to be C13H18O3 based on a quasi-
D-glucose which was identified by the method mentioned above
(Tanaka et al., 2007). The J value (7.9 Hz) of the anomeric proton
suggested the glucopyranosyl was also in a b configuration.
molecular ion peak at m/z 245.1149 (calcd for
C
13H18O3Na,
Accordingly, 9 was determined as b-D-glucopyranosyloxyl 9-oxo-
245.1148) in HRESIMS. Analysis of the 1H and 13C NMR spectra
(Tables 2 and 3) indicated that 7 had a structure similar with pte-
odonoic acid (11) (Cui et al., 1998), except for missing two olefinic
protons and two olefinic carbons. Comparison of the molecular
formulae and degrees of unsaturation in these two compounds
suggested that a carbonyl was connected to C-7 in 7 instead of
4,11(13)-eudesmadien-12-oate, and named alatoside G.
The 1H NMR spectrum (Table 3) of compound 10 displayed sig-
nals of two olefinic protons at dH 5.74 and 6.32, an anomeric proton
of sugar at dH 5.57 (J = 7.9 Hz), an oxygenated methine at dH 3.40, and
two methyl groups at dH 1.02 and 1.59. The 13C NMR spectrum (Table
2) showed resinances of four olefinic carbons (dC 134.6, 125.5, 146.3,

G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
205
125.0), one carboxyl group (dC 167.2), two methyl groups (dC 17.9,
19.2) and one glucose moiety (dC 95.9, 74.0, 78.8, 71.0, 78.1, 62.3).
These 1H and 13C NMR spectroscopic data were similar to those of
an isolated known compound 1b-hydroxy-eudesma-4,11(13)-
dien-12-oic acid (14) (Ohmoto et al., 1987), except for the presence
of an extra sugar moiety, suggesting that 10 was another eudesmane
glycoside. In addition, the correlationbetween H-10 (dH 5.57) and C-1
(dC 79.0) in HMBC spectrum indicated that the sugar moiety was
Sephadex LH-20 (Pharmacia, NJ, USA), respectively. All solvents
used in CC were of analytical grade (Tianjin Damao Chemical Plant,
Tianjin, P.R. China). Preparative high-performance liquid chroma-
tography (HPLC) was performed on a Varian chromatograph
equipped with a Prostar 215 pump, a Prostar 325 UV–VIS detector,
using a C18 reversed-phase column (Cosmosil, 20 ꢀ 250 mm, 5
lm).
4.2. Plant material
connected to C-1. Acid hydrolysis of 10 yielded D-glucose which
was identified by the method mentioned above (Tanaka et al.,
2007). The characteristic coupling constant of the anomeric proton
Whole plants of L. alata (D. Don) Sch. Bip. ex Oliv were collected
from Guangxi Medicinal Plant Garden in September, 2008, and
identified by Research Assistant You-Bao Huang of Guangxi Medic-
inal Plant Garden. A voucher specimen (No. 08091309) is deposited
in the Institute of Traditional Chinese Medicine and Natural Prod-
ucts, College of Pharmacy, Jinan University (Guangzhou, China).
(J = 7.9 Hz) indicated that it was a b-
D-glucoside. Therefore, 10 was
elucidated as 1b-O-(b- -glucopyranosyloxyl)-eudesma-4,11(13)-
D
dien-12-oic acid, and named alatoside H.
Recently, quite a few studies have focused on anticancer activi-
ties of sesquiterpenes, including eudesmane-type sesquiterpenes
(Cui et al., 2011; Dai et al., 2009; Minakawa et al., 2012; Tanaka
et al., 2012; Wang et al., 2007; Xu et al., 2010). The cytotoxic activi-
ties of some eudesmane-type sesquiterpene derivatives from L.
pterodonta were also investigated (Xiao et al., 2003). Therefore, the
present study tested the cytotoxic activities of all the isolated eudes-
mane-type sesquiterpenes with MTT assay on six human cancer cell
lines including MFC-7 (human breast carcinoma cell), A375 (human
melanoma cell), A549 (human lung carcinoma epithelial cell), Hep-2
(human larynx epidermoid carcinoma cell), CNE (human nasopha-
ryngeal carcinoma epithelia cell), and Hela (human cervical carci-
noma cell). However, all of the compounds were inactive on the
4.3. Extraction and isolation
The air-dried whole plants of L. alata (4.5 kg) were powdered
and percolated with EtOH–H2O (95:5, v/v) at room temperature
for three times. The extract was then concentrated in vacuo to yield
a residue (540 g), which was dissolved in EtOH–H2O (1:9, v/v) and
subjected to Diaion HP-20 macrosporous resin CC successively
eluted with EtOH–H2O mixtures (1:9, 3:7, 6:4, 9:1, v/v). The frac-
tion (70 g) eluted by EtOH–H2O (6:4, v/v) was concentrated and
then subjected to a silica gel CC (CHCl3–MeOH, 100:0 ? 50:50)
to afford 10 fractions. Fraction 2 (8.7 g) was separated by ODS silica
gel CC (MeOH–H2O, 15:85 ? 80:20) to give five subfractions
(2a–e). Subfraction 2b was purified by Sephadex LH-20 CC (CHCl3–
MeOH, 1:1) and preparative HPLC (MeOH–H2O, 70:30, v/v) to yield
compounds 1 (39 mg), 2 (28 mg), 3 (21 mg) and 19 (40 mg),
respectively. Compound 7 (25 mg) was recrystallized from subfrac-
tion 3c. Further purification of subfraction 3c by Sephadex LH-20
(CHCl3–MeOH, 1:1) and preparative HPLC (MeOH–H2O, 60:40,
v/v) afforded compounds 12 (28 mg), 13 (12 mg) and 14 (15 mg).
By similar procedures, compounds 11 (35 mg), 19 (29 mg) and 20
(18 mg) were obtained from subfraction 3d. Fraction 5 (5.9 g)
was subjected to silica gel CC using a petroleum ether (PE)–EtOAc
gradient as eluent, and then purified by preparative HPLC (MeOH–
H2O, 60:40, v/v) to give compounds 4 (21 mg), 5 (12 mg) and 15
(18 mg). Fraction 6 (6.1 g) was separated by a silica gel CC eluting
with gradient CHCl3–MeOH, and further purified by Sephadex LH-
20 (CHCl3–MeOH, 1:1) and preparative HPLC (MeOH–H2O, 50:50,
v/v) to afford compounds 6 (18 mg), 16 (16 mg) and 18 (24 mg),
respectively. Compounds 9 (14 mg), 21 (20 mg) and 22 (25 mg)
were isolated from fraction 7 (4.1 g) by ODS CC with a MeOH–
H2O gradient and preparative HPLC (MeOH–H2O (40:60, v/v)).
Fraction 9 (2.8 g) was purified by Sephadex LH-20 (CHCl3–MeOH,
1:1) and preparative HPLC (MeOH–H2O, 35:65, v/v) to give
compounds 8 (23 mg), 10 (16 mg) and 17 (34 mg).
tested cell lines in the concentration of 100 lg/mL.
3. Concluding remarks
Eudesmane sesquiterpenes are the primary chemical constitu-
ents of Laggera species, which is consistent with the results of this
study on L. alata. To the best of our knowledge, three cuauhtemone
derivatives had been previously reported from L. alata (Zdero and
Bohlmann, 1989), and this paper added 22 cuauhtemone derivatives
into the current list of the compounds isolated from the plant. More-
over, there were no such compounds reported to be isolated from
L. pterodonta, another Laggera plant found in China. This was helpful
in chemotaxonomical classifications. It is noteworthy that the ste-
reochemistry of the cuauhtemone derivatives determined in this pa-
per by single crystal X-ray diffraction analysis and modified
Mosher’s method is consistent with that proposed by Nakanishi
et al. (1974), rather than that described by Torres-Valencia et al.
(2003). Quite a few reports concerned the anticancer activities of
eudesmane-type sesquiterpenes (Cui et al., 2011; Dai et al., 2009;
Minakawa et al., 2012; Tanaka et al., 2012; Xu et al., 2010); however,
in the present study, the eudesmane sesquiterpenes isolated from L.
alata possessed no cytotoxic effects on human cancer cell lines.
4. Experimental
4.3.1. (3R,4S,5R,6R,20R,30S)-3-O-(20,30-Diacetoxy-20-methylbutyryl)-
4.1. General experimental procedures
cuauhtemone (1)
Colorless blocks; mp 118–120 °C, ½a D25
ꢁ
+21.6 (c 0.2, MeOH); UV
(MeOH) kmax: 254 nm; IR (KBr) mmax: 3473, 2942, 2867, 1739, 1449,
Melting points were obtained on an X-5 micro-melting point
apparatus. Optical rotations were recorded on a JASCO P-1020
polarimeter, whereas IR spectra were measured on a JASCO FT/IR-
480 plus infrared spectrometer with KBr pellets. UV spectra were
acquired on a JASCO V-550 UV/VIS spectrometer. HRESIMS data
were determined on an Agilent 6210 ESI/TOF mass spectrometer.
1D NMR (1H, 13C, and DEPT) and 2D (1H–1H COSY, HSQC, HMBC,
and ROESY) NMR spectra were recorded on a Bruker Avance 400
or 500 NMR spectrometer with TMS as internal standard, and chem-
ical shifts were expressed in d values (ppm). Column chromatogra-
phy (CC) employed silica gel (200–300 mesh; Qingdao Marine
Chemical Inc., Qingdao, P.R. China), ODS (YMC, Kyoto, Japan) and
1372, 1197, 1143, 952, 607, 526 cmꢂ1; for 1H NMR (CDCl3,
500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic data, see
Tables 1 and 2; HRESIMS m/z: 475.2310 [M+Na]+ (calcd for
C24H26O8Na, 475.2302).
4.3.2. 4-O-Acetyl-3-O-(30-acetoxy-20-hydroxy-20-methylbutyryl)-
cuauhtemone (2)
Brown oil, ½a 2D5
ꢁ
+21.0 (c 0.2, MeOH); UV (MeOH) kmax: 254 nm;
IR (KBr) mmax: 3475, 2939, 2867, 1736, 1448, 1375, 1195, 1145, 942,
615 cmꢂ1; for 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,

206
G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
125 MHz) spectroscopic data, see Tables 1 and 2; HRESIMS m/z
3; HRESIMS m/z 435.1981 [M+Na]+ (calcd for C21H30O8Na,
435.1989).
475.2302 [M+Na]+ (calcd for C24H26O8Na, 475.2302).
4.3.3. 4-O-Acetyl-3-O-(20,30-diacetoxy-20-hydroxy-20-methylbutyryl)-
4.4. X-ray data of 1 and 7
cuauhtemone (3)
Brown oil, ½a 2D5
ꢁ
+21.7 (c 0.2, MeOH); UV (MeOH) kmax: 254 nm;
4.4.1. X-ray data of cuauhtemone A (1)
Colorless blocks, C24H36O8, Mr = 452.53, orthorhombic, space
group P212121, a = 8.24486(12) Å, b = 10.30986(18) Å, c = 28.5017
(5) Å, V = 2422.74(7) Å3, Z = 4, dx = 1.241 Mg/m3, F(000) = 976,
IR (KBr) mmax: 3473, 2942, 2837, 1740, 1448, 1372, 1242, 1139,
1016, 949, 674 cmꢂ1; for 1H NMR (CDCl3, 400 MHz) and 13C NMR
(CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; HRESIMS
m/z 517.2406 [M+Na]+ (calcd for C26H38O9Na, 517.2408).
l
(CuKa
) = 0.762 mmꢂ1. Data collection was performed on a Gemini
S Ultra using graphite-monochromated radiation (k = 1.54184 Å);
4.3.4. 3-O-(30-Acetoxy-20-hydroxy-20-methylbutyryl)-cuauhtemone
3843 unique reflections were collected to hmax = 62.77°, where
3652 reflections were observed [F2 > 2 (F2)]. The structures were
r
(4)
Brown oil, ½a 2D5
ꢁ
+23.2 (c 0.2, MeOH); UV (MeOH) kmax: 254 nm;
solved by direct methods (SHELXS 97) and refined by full-matrix
least-squares on F2. The final R = 0.0340, Rw = 0.0895, and S = 1.027.
IR (KBr) mmax: 3464, 2941, 2828, 1735, 1453, 1374, 1264, 1200,
1143, 990, 952, 851, 672 cmꢂ1; for 1H NMR (CDCl3, 400 MHz)
and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1
and 2; HRESIMS m/z 433.2191 [M+Na]+ (calcd for C22H34O7Na,
433.2197).
4.4.2. X-ray data of 3-oxo-di-nor-eudesma-4-en-11-oic acid (7)
Colorless blocks, C13H18O3, Mr = 222.27, monoclinic, space group
P21, a = 7.6924(4) Å, b = 17.6450(8) Å, c = 9.0224(4) Å, V = 1179.05
(10) Å3,
Z = 4,
dx = 1.252 Mg/m3,
F(000) = 480,
l(CuKa) =
0.710 mmꢂ1. Data collection was performed on a Gemini S Ultra
using graphite-monochromated radiation (k = 1.54184 Å); 2894 un-
ique reflections were collected to hmax = 60.88°, where 2590 reflec-
4.3.5. 4-O-Acetyl-3-O-(20,30-dihydroxy-20-methylbutyryl)-
cuauhtemone (5)
Brown oil, ½a 2D5
ꢁ
+24.7 (c 0.2, MeOH); UV (MeOH) kmax: 254 nm;
IR (KBr) mmax: 3503, 2929, 2860, 1729, 1461, 1379, 1270, 1171,
tions were observed [F2 > 2 (F2)]. The structures were solved by
r
direct methods (SHELXS 97) and refined by full-matrix least-squares
1115, 989, 731, 675 cmꢂ1; for 1H NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2;
HRESIMS m/z 433.2195 [M+Na]+ (calcd for C22H34O7Na, 433.2197).
on F2. The final R = 0.0460, Rw = 0.1185, and S = 1.050.
The above crystallographic data have been deposited at the
Cambridge Crystallographic Data Center under CCDC 909109 for
cuauhtemone A (1) and CCDC 909110 for 3-oxo-di-nor-eudesma-
4-en-11-oic acid (7). These data can be obtained free of charge
via www.ccdc.cam.ac.uk/deposit (or from the CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
4.3.6. 4a-Acetoxy-3a
-(30-acetoxy-20-methylbutyryloxy)-11-hydroxy-
6,7-dehydroeudesman-8-one (6)
Brown oil, ½a 2D5
ꢁ
+23.4 (c 0.2, MeOH), UV (MeOH) kmax: 254 nm;
IR (KBr) mmax: 3449, 2954, 1744, 1638, 1459, 1376, 1239, 1134,
1105, 951, 873, 607 cmꢂ1; for 1H NMR (CDCl3, 400 MHz) and 13C
NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2;
HRESIMS m/z 491.2242 [M+Na]+ (calcd for C24H36O9Na, 491.2251).
4.5. Acid hydrolysis and HPLC analysis of 8–10
Compounds 8–10 (each 2.0 mg) were dissolved in 2 N HCl
(10 ml) and heated at 80 °C for 5 h, respectively. The aglycone
was extracted with CH2Cl2, and the aqueous layer was dried by
N2. Dry pyridine (0.5 ml) and L-cysteine methyl ester hydrochloride
(2.0 mg) were added to the residue. The mixture was heated at
60 °C for 1 h and then dried by N2. O-Toyl isothiocyomate
(3–5 ll) was added, and the mixture was heated at 60 °C for 1 h.
Then, the reaction mixture was directly analyzed by standard C18
4.3.7. 3-Oxo-di-nor-eudesma-4-en-11-oic acid (7)
Colorless blocks, mp 120–122 °C, ½a D25
ꢁ
+23.8 (c 0.2, MeOH); UV
(MeOH) kmax: 254 nm; IR (KBr)
mmax: 3449, 2954, 1744, 1638,
1459, 1376, 1239, 1134, 1105, 950, 872, 607 cmꢂ1; for 1H NMR
(CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic
data, see Tables 2 and 3; HRESIMS m/z 245.1149 [M+Na]+ (calcd
for C13H18O3Na, 245.1148).
HPLC and detected by a UV detector [column: Capcell pak C18
4.3.8. Alatoside F (8)
(4.6 ꢀ 250 mm, 5
lm); mobile phase: water (0.1% HCO2H)-CH3CN
Colorless oil, ½a D25
ꢁ
+26.2 (c 0.2, MeOH), UV (MeOH) kmax:
(75:25); follow rate: 0.8 mL/min, column temperature: 25 °C;
detector: VWD; detector wavelength: 250 nm]. The standard sug-
ars were treated by the same reaction and chromatographic condi-
254 nm; IR (KBr) mmax: 3419, 2929, 1715, 1650, 1453, 1402, 1384,
1332, 1274.1, 1244, 1155, 1079, 674 cmꢂ1; for 1H NMR (CD3OD,
500 MHz) and 13C NMR (CD3OD, 125 MHz) spectroscopic data,
see Tables 2 and 3; HRESIMS m/z 433.1831 [M+Na]+ (calcd for
tions. As a result, D-glucose was detected from the hydrolyzate of
compounds 8–10 with the same retention time of standard sugar
derivatives, respectively.
C21H30O8Na, 433.1833).
4.3.9. Alatoside G (9)
4.6. Preparation of (R)- and (S)-MTPA esters of 22
Colorless oil, ½a D25
ꢁ
+20.8 (c 0.2, MeOH), UV (MeOH) kmax:
254 nm; IR (KBr) mmax: 3421, 2933, 1711, 1631, 1453, 1401, 1384,
Compound 1 (10 mg) was dissolved in 2 N HCl (10 ml) and
heated at 80 °C for 5 h. The solution was extracted with CH2Cl2
and then concentrated in vacuo to afford the aglycone, which
was identical to the isolated compound 22 by comparison of their
NMR and optical rotations. Compound 22 was dissolved in deuter-
ated pyridine (0.5 ml) in a clean NMR tube under a gentle N2
stream and a 1H NMR spectrum was recorded as a reference. Then
1252, 1076, 708, 601 cmꢂ1; for 1H NMR (CD3OD, 500 MHz) and
13C NMR (CD3OD, 125 MHz) spectroscopic data, see Tables 2 and
3; HRESIMS m/z 433.1835 [M+Na]+ (calcd for C21H30O8Na,
433.1833).
4.3.10. Alatoside H (10)
Colorless oil, ½a D25
ꢁ
+20.8 (c 0.2, MeOH), UV (MeOH) kmax
:
(S)-a-methoxy-a-(trifluoromethyl) phenylacetic chloride (5 lL)
254 nm; IR (KBr)
m
max: 3418, 2933, 1722, 1676, 1406, 1245,
was added into the NMR tube under the N2 gas stream and imme-
diately shaken until uniformly mixed. After sealing with parafilm,
the reaction NMR tube was kept for 8 h at room temperature.
1076.4, 956, 873, 607 cmꢂ1; for 1H NMR (CD3OD, 500 MHz) and
13C NMR (CD3OD, 125 MHz) spectroscopic data, see Tables 2 and

G.-C. Wang et al. / Phytochemistry 96 (2013) 201–207
207
The 1H NMR spectrum, recorded directly from the reaction NMR
tube, showed the production of the corresponding (R)-MTPA ester
(22a). In an identical fashion, another portion of compound 22
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5. MTT assay
Evaluation of the cytotoxic activities of all the isolated com-
pounds on MFC-7, A375, A549, Hep-2, CNE, and Hela cells was car-
ried out as described previously (Wu et al., 2012a,b). All the cells
were purchased from the Cell Bank of Type Culture Collection of
the Chinese Academy of Sciences (Shanghai, China).
Acknowledgments
This work was supported by Grants from the Fundamental
Research Funds for the Central Universities (21612417), the Na-
tional Natural Science Foundation (Nos. 81072535, 81273390),
and Research Team Program of Natural Science Foundation of
Guangdong Province (No. 8351063201000003).
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Appendix A. Supplementary data
Torres-Valencia, J.M., Quintero-Mogica, D.L., León, G.I., Suárez-Castillo, O.R.,
Villagómez-Ibarra, J.R., Maldonado, E., Cerda-García-Rojas, C.M., Joseph-
Nathan, P., 2003. The absolute configuration of cuauhtemone and related
compounds. Tetrahedron: Asymmetry 14, 543–548.
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Pluchea sagittalis. Their antifeedant activity on Spodoptera frugiperda.
Phytochemistry 69, 1689–1694.
Wang, C.M., Jia, Z.J., Zheng, R.L., 2007. The effect of 17 sesquiterpenes on cell
viability and telomerase activity in the human ovarian cancer cell line HO-8910.
Planta Med. 73, 180–184.
Wu, X., Wang, L., Wang, G.C., Wang, H., Dai, Y., Ye, W.C., Li, Y.L., 2012a. Steroidal
saponins and sterol glycosides from Paris polyphylla var. yunnanensis. Planta
Med. 78, 1–9.
Wu, X., Wang, L., Wang, H., Dai, Y., Ye, W.C., Li, Y.L., 2012b. Steroidal saponins from
Paris polyphylla var. yunnanensis. Phytochemistry 81, 133–143.
Xiao, Y.C., Zheng, Q.X., Zhang, Q.J., Sun, H.D., Guéritte, F., Zhao, Y., 2003. Eudesmane
derivatives from Laggera pterodonta. Fitoterapia 74, 459–463.
Xu, X.Y., Xie, H.H., Hao, J., Jiang, Y.M., Wei, X.Y., 2010. Eudesmane sesquiterpene
glucosides from lychee seed and their cytotoxic activity. Food Chem. 123, 1123–
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2013.
07.014.
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