Structural investigation and biological activity of sesquiterpene lactones from the Traditional Chinese Herb Inula racemosa

Article abstract of DOI:10.1021/np300742d

Five new sesquiterpene lactones, racemosalactones A-E (1-5), along with 19 known sesquiterpene latones (6-24), were isolated from the roots of Inula racemosa. Their structures were elucidated by extensive spectroscopic analysis, and the absolute configuration of 2 was deduced from X-ray diffraction analysis. Compounds 1, 6, 8, 10, 12, 14, and 17 exhibited antiproliferative activities with IC₅₀ values ranging from 0.38 to 4.19 μg/mL against human non-small-cell lung cancer A549, hepatocellular carcinoma HepG2, and human fibrosarcoma HT1080 cells. Compounds 6 and 8 exhibited antiproliferative activities against endothelial cells with IC₅₀ values of 2.4 and 2.5 μg/mL, respectively. Furthermore, compounds 6 and 8 both inhibited endothelial cell tube formation at 1.0 μg/mL. A method for the rapid and straightforward preparative-scale isolation of compound 6 from alantolides is described.

Full text of DOI:10.1021/np300742d

Article
pubs.acs.org/jnp
Structural Investigation and Biological Activity of Sesquiterpene
Lactones from the Traditional Chinese Herb Inula racemosa
Yan-Yan Ma, Deng-Gao Zhao, and Kun Gao*
State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou
730000, People’s Republic of China
*
S Supporting Information
ABSTRACT: Five new sesquiterpene lactones, racemosalac-
tones A−E (1−5), along with 19 known sesquiterpene latones
(
6−24), were isolated from the roots of Inula racemosa. Their
structures were elucidated by extensive spectroscopic analysis,
and the absolute configuration of 2 was deduced from X-ray diffraction analysis. Compounds 1, 6, 8, 10, 12, 14, and 17 exhibited
antiproliferative activities with IC50 values ranging from 0.38 to 4.19 μg/mL against human non-small-cell lung cancer A549,
hepatocellular carcinoma HepG2, and human fibrosarcoma HT1080 cells. Compounds 6 and 8 exhibited antiproliferative
activities against endothelial cells with IC50 values of 2.4 and 2.5 μg/mL, respectively. Furthermore, compounds 6 and 8 both
inhibited endothelial cell tube formation at 1.0 μg/mL. A method for the rapid and straightforward preparative-scale isolation of
compound 6 from alantolides is described.
hytochemical investigations of the Asteraceae (Composi-
P
tae) family for structurally diverse and biologically
significant sesquiterpenoids have been attractive programs of
1
2
natural products and synthesis chemistry. The genus Inula
comprises about 100 species and is distributed widely in Asia,
3
Europe, and Africa. Inula racemosa Hook.f. is an economically
and medicinally important perennial herb that has long been
used in China, India, and Europe. It is a rich source of
sesquiterpene lactones, a widely distributed class of natural
products with interesting biological activities. Previous studies
have shown that the roots of I. racemosa contain high levels of
3,4
eudesmane-type sesquiterpene lactones, in particular alanto-
5
lactone (6) and isoalantolactone (8). Pharmacological studies
showed that 6, 8, and their analogues possess a wide spectrum
of biological activities, such as antitumor, antifungal, anti-
6
bacterial, insecticidal, and plant growth regulatory activities. As
a part of our continuous search for novel sesquiterpene lactones
from plants of the Asteraceae family, and in an attempt to learn
more about the structure and biological activity relationships
among the sesquiterpene lactones of I. racemosa, four
eudesmane-type (1−4) and one eremophilane-type (5)
sesquiterpene lactone, together with 19 known analogues (6−
4a
7
alantolactone (8), 11,13-dihydroisoalantolactone (9), alloa-
4a
6b
lantolactone (10), 3-oxoalloalantolactone (11), 5α-epox-
4b
4c
yalantolactone (12), dihydroepoxyalantolactone (13),
(15)α-epoxyisoalantolactone (14), dihydro-4(15)α-epoxyi-
soalantolactone (15), telekin (16), isotelekin (17),
macrophyllilactone E (18), 3β-hydroxy-11α,13-dihydroalanto-
lactone (19), 11α-hydroxyeudesm-5-en-8β,12-olide (20),
1,13-dihydro-2α-hydroxyalantolactone (21), 11,13-dihydroi-
valin (22), 11βH-2α-hydroxyeudesman-4(15)-en-12,8β-olide
23), and 11,12,13-trinoreudesm-5-ene-7β,8α-diol (24).
Racemosalactone A (1) was obtained as colorless crystals
2
4), were isolated from the roots of I. racemosa. Herein, we
4b
4
report the structural elucidation and biological activity of the
sesquiterpene lactones from the roots of I. racemosa.
6b 4d 4a
8
3
3
9
RESULTS AND DISCUSSION
1
■
10
The MeOH extract of the roots of I. racemosa was partitioned
11
12
(
into H O and extracted with petroleum ether, EtOAc, and n-
2
BuOH. The petroleum ether fraction was subjected to repeated
column chromatography over silica gel, Sephadex LH-20, and
ODS to afford five new sesquiterpene lactones, racemosalac-
tones A−E (1−5). The 19 known compounds were identified
after crystallization from CHCl . Its HRESIMS exhibited an [M
3
Received: October 25, 2012
4a
7
as alantolactone (6), 11,13-dihydroalantolactone (7), iso-
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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1
Table 1. H NMR Data of Compounds 1−5 (400 MHz, δ in ppm, J in Hz)
a
b
a
a
a
position
1
1
2
3
4
5
1.17, m
.50, m
1.41, m
.70, m
1.95, m
.41, m
1.21, ddd (13.2, 13.2, 3.2)
1.66, ddd (13.2, 3.2, 3.2)
1.60, m
1.12, m
1.68, m
1.45, m
1.83, m
1.57, m
1.57, m
2.52, m
1.81, d (12.0)
5.61, d (4.4)
1
1.90, ddd (12.0, 4.0, 1.6)
2
3
1
1.84, m
3.84, m
4.11, m
1.73, m
1.69, m
1.79, m
3.67, m
1.99, dd (12.0, 12.0)
2.68, ddd (12.0, 4.0, 1.6)
1
4
5
6
1.70, m
2.63, m
1.81, d (12.4)
1.41, m
5.42, d (3.2)
5.29, s
1.63, ddd (13.6, 6.0, 2.0)
1.05, ddd (13.6, 12.4, 12.4)
2.29, m
1.64, m
1.58, m
2.05, m
4.64, m
2.38, m
1
.86, dd (14.0, 12.0)
7
8
9
3.36, ddd (12.0, 6.0, 6.0)
4.55, m
2.97, dd (5.2, 3.2)
5.07, ddd (5.2, 3.2, 2.4)
1.55, m
4.53, m
1.63, m
4.96, brs
1.70, m
1.50, dd (13.2, 4.4)
2.25, m
2
.03, dd (15.2, 5.2)
2.17, dd (15.2, 3.2)
2.03, dd (15.2, 2.8)
2.90, m
2.61, dd (12.8, 7.6)
2.33, m
1
1
1
3
6.12, d (1.2)
.54, d (1.2)
1.15, s
3.85, d (12.4)
3.61, d (12.4)
1.19, s
1.28, d (7.6)
5
1.22, d (7.6)
1.21, s
1.46, s
0.79, s
4.90, s
4.57, s
1
1
4
5
0.91, s
1
.03, d (7.6)
1.08, d (7.2)
1.14, d (7.6)
0.91, d (6.4)
a
b
Recorded in CDCl . Recorded in methanol-d .
3
4
Table 2. 13C NMR Data of Compounds 1−5 (100 MHz, δ in ppm)
a
b
a
a
a
position
1
2
3
4
5
1
2
3
4
5
6
7
8
9
35.3, CH2
28.1, CH2
16.2, CH2
41.0, CH
74.7, C
41.1, CH2
26.0, CH2
73.5, CH
47.0, CH
153.0, C
16.7, CH2
32.6, CH2
41.9, CH2
38.6, CH
154.6, C
117.3, CH
73.8, C
50.8, CH2
67.1, CH
46.2, CH2
146.1, C
125.6, CH
63.6, CH
36.0, CH2
30.7, CH
37.6, C
45.8, CH
21.1, CH2
46.0, CH
77.0, CH
40.9, CH2
34.0, C
36.1, CH
37.8, CH
77.4, CH
39.3, CH2
35.4, C
117.0, CH
46.6, CH
78.3, CH
43.3, CH2
33.4, C
36.7, CH2
39.7, CH
78.9, CH
34.5, CH2
144.0, C
82.0, CH
39.6, CH2
33.7, C
1
1
1
1
1
1
0
1
2
3
4
5
142.2, C
79.8, C
46.6, CH
176.1, C
8.4, CH3
28.3, CH3
22.8, CH3
77.7, CH
177.0, C
40.5, CH
179.9, C
170.8, C
176.4, C
120.0, CH2
23.2, CH3
16.7, CH3
47.1, CH2
29.2, CH3
16.2, CH3
19.1, CH3
18.8, CH3
109.0, CH3
15.7, CH3
19.1, CH3
15.5, CH3
a
b
Recorded in CDCl . Recorded in methanol-d .
3
4
+
+
H − H O] peak at m/z 233.1531 and established the
deficiency identified compound 1 as a tricyclic compound. The
H− H COSY spectrum indicated the H-1/H-2/H-3/H-4/H-
2
1
1
molecular formula C H O , implying an index of hydrogen
deficiency of five. The IR spectrum suggested the presence of
hydroxy (3439 cm ) and α-methylene-γ-lactone functionalities
1733 and 1664 cm ). The H NMR data (Table 1) showed
two characteristic α-methylene-γ-lactone doublets at δH 6.12
1H, d, J = 1.2 Hz) and 5.54 (1H, d, J = 1.2 Hz), respectively.
Two methyl resonances were evident: a singlet at δ 1.15 (3H,
s) and a doublet at δ 1.03 (3H, d, J = 7.6 Hz). The C NMR
data showed 15 carbon signals (Table 2), which were assigned
1
5
22
3
1
5 and H-6/H-7/H-8/H-9 proton sequences. The HMBC
−1
cross-peaks between H-13/C-7, H-13/C-12, and H-6/C-8
indicated that a γ-lactone function was formed between C-8
and C-11. The HMBC correlations from H-14 to C-1, C-5, C-
−1
1
(
(
9
, and C-10 confirmed that Me-14 was connected to C-10, and
H
correlations from H-15 to C-3, C-4, and C-5 confirmed that
Me-15 was connected to C-4. These correlations (Figure 1)
established the molecular skeleton of 1 as a eudesmane-type
sesquiterpene lactone. The placement of the C-5 hydroxy group
was confirmed from the HMBC correlations of the angular
13
H
2
3
by a DEPT experiment as two sp and two sp quaternary
carbons (including one oxygenated), three sp methines
including one oxygenated), one sp and five sp methylenes,
3
2
3
(
methyl (δ 1.15, H-14) and methyl doublet (δ 1.03, H-15)
and two methyls. The lactone carbonyl and olefinic resonances
H
H
in the 13C NMR data (Table 2) appeared at δ 170.8 (C-12),
C
with the oxygenated quaternary carbon resonating at δ 74.7
C
1
42.2 (C-11), and 120.0 (C-13), respectively, revealing the
(C-5). Thus, the molecular framework of 1 was established.
The relative configuration of 1 was determined by X-ray single-
crystal diffraction studies (Figure 2). Consequently, the
presence of a methylene group conjugated with a lactone
carbonyl group. Therefore, the remaining index of hydrogen
B
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Journal of Natural Products
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−1
−1
and 3268 cm ) and γ-lactone (1748 cm ) groups. The NMR
data (Tables 1 and 2) exhibited signals typical of a eudesmane-
type sesquiterpene lactone including an ester carbonyl (δC
1
1
76.4, C-12), a trisubstituted carbon−carbon double bond [δ
C
53.0 (C-5), 117.0 (C-6); δ 5.42 (d, J = 3.2 Hz, H-6)], and a
H
CH Cl group [δ 47.1 (C-13); δ 3.85, 3.61 (1H each, d, J =
2
C
H
1
2.4 Hz)]. The CH Cl group was connected to C-11, as
2
indicated by the HMBC correlations from H-13 (δ 3.85, 3.61)
H
to C-12 (δ 176.4), C-11 (δ 79.8), and C-7 (δ 46.6). The H-
C
C
C
1
/H-2/H-3/H-4/H-15 and H-6/H-7/H-8/H-9 proton spin
1
1
systems were deduced from the H− H COSY correlations.
The HMBC spectrum showed the following correlations: H-1
with C-3; H-6 with C-8; H-7 with C-12; H-13 with C-11 and
C-12; H-14 with C-1, C-5, and C-9; and H-15 with C-3, C-4,
and C-5. These data (Figure 1) established the molecular
skeleton of 2. The relative configuration of 2 was determined
by X-ray crystallographic analysis (Figure 2). The presence of
the chlorine atom in 2 and the value of the Flack parameter
[
−0.02(7)] permitted the determination of the absolute
13
configuration of 2. In this way, the structure of 2 was defined
as (3S,4R,7S,8R,10R,11R)-3,11-dihydroxyeudesm-5-en-12,8-
olide.
Figure 1. Key 2D NMR correlations of compounds 1−5.
Racemosalactone C (3) was obtained as a colorless oil and
exhibited a pseudomolecular ion peak at m/z 251.1642 [M +
+
structure of 1 was determined as 5α-hydroxyeudesm-11(13)-
en-12,8β-olide.
Racemosalactone B (2) was obtained as colorless, flaky
crystals after crystallization from MeOH. The presence of a
chloride atom was evident from a series of characteristic
H] under HRESIMS, which is consistent with a molecular
formula of C15H O . The IR spectrum suggested the presence
22 3
−
1
−1
of hydroxy (3448 cm ) and carbonyl (1749 cm ) groups. The
NMR data indicated that 3 was also a eudesmane-type
sesquiterpene lactone similar to the known compound 7,
except that it had one hydroxy group not present in 7. The
presence of the C-7 hydroxy group was confirmed from the
isotopic peaks in the ESIMS, with a ratio of 3:1 at m/z 301/303
+
and 318/320. Its HRESIMS showed an [M + NH ] signal at
4
m/z 318.1467, suggesting a molecular formula of C H ClO .
HMBC (Figure 1) correlations from H-9 (δ 2.03, dd, J = 15.2,
1
5
21
4
H
The IR spectrum showed absorption bands for hydroxy (3463
2.8 Hz) and H-13 (δ 1.22, d, J = 7.6 Hz) to C-7 (δ 73.8). An
H
C
Figure 2. X-ray crystal structure of compounds 1, 2, 4, and 5.
C
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Journal of Natural Products
1,13-dihydroalantolactone skeleton was determined using the
H− H COSY cross-signals for H-1/H-2/H-3/H-4/H-15, H-6/
H-7/H-8/H-9, and H-11/H-13 coupling systems and the
following HMBC correlations: H-6 with C-8 and C-10; H-13
with C-7 and C-12; H-14 with C-1, C-5, C-9, and C-10; and H-
Article
6
5
1
assays. It is also a major constituent of I. racemosa. However,
previous teams have only been able to isolate 6 either at low
levels of purity or after several tedious sequential purification
1
1
4
,5,17
steps.
As previously reported, we obtained large amounts
of alantolides (mixture of 6 and 8, 153 g) and only small
amounts of 6 (20 mg). For this reason, we created a method
that was amenable to the rapid and straightforward preparative-
scale isolation of 6 from alantolides. This method involves a
15 with C-3, C-4, and C-5.
The relative configuration of 3 was deduced from the
ROESY spectrum and biogenetic consideration. ROESY
correlations (Figure 1) of H-15/H-14 and H-14/H-13
indicated that Me-13, Me-14, and Me-15 were β-oriented.
The cross-peaks of H-11 and H-8 revealed that they were α-
silica gel column but does not require specialized equipment
4
such as silica gel LC columns impregnated with AgNO
. As
3
described in the Experimental Section, the alantolides were
treated with selenium dioxide and tert-butyl hydroperoxide,
which selectively oxidized C-3 of 8 to produce 17 while 6
remained intact (Scheme 1). This process occurred with
14
oriented. On the basis of biogenetic considerations, the C-7
hydroxy group was predicted to be α-oriented. In this way,
compound 3 was identified as 7α-hydroxyeudesm-5-en-12,8β-
olide.
a
Racemosalactone D (4) was obtained as colorless crystals
Scheme 1
after crystallization from CHCl . The HRESIMS exhibited an
3
+
[
M + NH ] peak at m/z 284.1856 and established a molecular
4
formula of C H O . The IR spectrum showed absorption
bands of hydroxy (3492 and 3320 cm ), γ-lactone (1770
cm ), and olefinic (1649 cm ) groups. The NMR data of 4
(
1
5
22
4
−1
−1
−1
10
Tables 1 and 2) were similar to those of 22, expect that a
a
Reagents and condition: (a) SeO /t-BuOOH, CH Cl , 90%.
2
2
2
methine carbon (δC 39.9, C-11) in 22 was replaced by an
oxygenated quaternary carbon (δ 77.4, C-11) in 4, as indicated
C
by the HMBC correlations from H-13 to C-7, C-11, and C-12
excellent regio- and stereoselectivity. Compounds 6 and 17
could subsequently be separated using column chromatography
over silica gel. In this way, the present method showed superior
isolation efficiency to previous methods.
(
Figure 1). The connectivity was further established by means
1
1
of H− H COSY and HMBC experiments (Figure 1). The
relative configuration of compound 4 was determined by X-ray
single-crystal diffraction studies (Figure 2). Thus, the structure
of compound 4 was established as 2α,11α-dihydroxyeudesm-4-
en-12,8β-olide.
The n-hexane fraction of I. racemosa exhibits promising
5
antiproliferative activity. The antiproliferative activity of some
10,18
previously isolated compounds has also been investigated.
Racemosalactone E (5) was obtained as colorless crystals
All sesquiterpene lactones obtained here were screened for
their antiproliferative activity against human non-small-cell lung
cancer A549, hepatocellular carcinoma HepG2, and human
fibrosarcoma HT1080 cells (Table 3) to identify the functional
groups necessary for maintaining and increasing its activity.
This may lead to the development of more effective antitumor
agents. It is widely believed that α,β-unsaturated carbonyl
compounds, particularly α-methylene lactones, have significant
antiproliferative activity. Alkylation of biological nucleophiles
by α,β-unsaturated carbonyl structures in Michael-type addition
after crystallization from CHCl . Its molecular formula,
3
C H O , was established using the HRESIMS spectrum (m/
1
5
22
3
+
z 268.1912, [M + NH ] ), implying an index of hydrogen
4
deficiency of five. The IR absorptions were observed at 3511
−1
and 1742 cm , suggesting the presence of hydroxy and
1
3
carbonyl groups. The C NMR spectrum showed 15 carbon
signals, which were assigned by a DEPT experiment to three
methyl, three methylene, six methine (two oxymethine), and
three quaternary carbons. The above-mentioned and the
chemical shifts and splitting pattern of the resonances at δH
1
9,20
is considered the general mechanism of action.
Our results
1
.28, 0.90, and 0.92 (H -13, H -14, H -15, respectively) in the
were consistent with this conclusion (Table 3). The absence of
an α-methylene-γ-lactone moiety diminished the antiprolifer-
ative activities of these sesquiterpene lactones, as shown by
comparisons of 7 with 6, 9 with 8, 11 with 10, 13 with 12, and
14 with 15.
3
3
3
1
H NMR spectrum indicated an eremophilanolide skeleton for
5
15
. The NMR data (Tables 1 and 2) showed the appearance of
one trisubstituted double bond [δ 144.0 (C-10), 125.6 (C-1);
C
δ 5.61 (d, J = 4.4 Hz, H-1)] and one ester carbonyl (δ 179.9,
H
C
C-12). The presence of the three segments CH(1)−CH(2)−
Among these α-methylene lactones, 6 and 8 showed the
CH (3)−CH (4)−CH (15), CH (6)−CH (7)−CH(8)−
most potent antiproliferative activities, with IC values ranging
2
3
2
50
CH (9), and CH(7)−CH(11)−CH (13) was inferred from
from 0.38 to 1.77 μg/mL. When 16 and 17 were compared to
8, the antiproliferative activities were reduced because of the
hydroxy groups at C-5 in 16 and C-3 in 17. Oxidation of the C-
5−C-6 olefinic bond in 6 or the C-4−C-15 olefinic bond in 8 to
an oxirane moiety, as in 12 or 14, significantly reduced
antiproliferative activity. Compounds 1 and 10 showed
significantly less antiproliferative activity than compound 6
because of the hydroxy group at C-5 in 1 and the C-4−C-5
olefinic bond in 10. These structure−activity relationships
suggested that the relative lipophilicities of these α-methylene
lactones may contribute to the activity.
2
3
1
1
the H− H COSY spectrum, which showed the H-1/H-2/H-3/
H-4/H-15, H-6/H-7/H-8/H-9, and H-7/H-11/H-13 coupling
systems. The HMBC correlations of H-9 with C-1 and C-5; H-
1
3 with C-7, C-11, and C-12; H-14 with C-5, C-6, and C-10;
and H-15 with C-3, C-4, and C-5 established the molecular
framework of 5 (Figure 1). The chemical shifts of C-1, C-2, and
16
C-3 in 5 were similar to those of armatin B, suggesting that
the C-2 hydroxy group was α-oriented. The structure of 5 was
thus defined as 2α-hydroxyeremophil-1(10)-en-8β,12-olide. An
X-ray diffraction analysis corroborated the proposed structure
(
Figure 2).
Inhibition of angiogenesis (antiangiogenesis) is an effective
strategy in the treatment of human cancer. Thus, we evaluated
the antiproliferative activity of the isolated compounds against
Alantolactone (6) possesses a wide spectrum of biological
activities and exhibits strong activities in those biological
D
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Journal of Natural Products
Article
Table 3. IC50 Values for Antiproliferative Activity of
Compounds 1−24
cell type (IC , μg/mL)
5
0
compound
A549
HepG2
HT1080
mitomycin
0.54
1.51
37.32
27.76
>50
0.14
2.76
>50
>50
>50
>50
1.3
0.15
1.88
>50
1
2
3
4
5
6
7
8
9
>50
>50
38.58
0.55
24.79
0.38
>50
>50
0.696
>50
>50
1.77
>50
3.57
>50
2.73
25.3
3.78
>50
8.14
4.19
31.2
>50
>50
26.11
>50
>50
>50
0.795
>50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2.67
22.54
1.59
16.23
2.65
>50
3.87
>50
1.32
17.41
3.51
>50
4.27
1.47
16.22
>50
3.5
2.44
28.51
>50
>50
>50
24.8
>50
20.1
>50
>50
>50
>50
>50
human umbilical vein endothelial cells (HUVECs). Com-
pounds 6 and 8 exhibited antiproliferative activity against
HUVECs, showing IC50 values of 2.4 and 2.5 μg/mL,
respectively. As shown in Figure 3, they both inhibited
endothelial cell tube formation at 1.0 μg/mL. These data
suggest that the antiproliferative activities of compounds 6 and
Figure 3. Effects of compounds 6 and 8 on in vitro matrigel
angiogenesis assays. Colchicine was used as a positive control.
8
are not more pronounced in endothelial cells than in tumor
cells.
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
determined on an X-4 digital display micromelting point apparatus
and are uncorrected. Optical rotations were measured on a Perkin−
Elmer 341 polarimeter with 1 dm cell. IR spectra were obtained on a
Nicolet NEXUS 670 FT-IR spectrometer. NMR spectra were recorded
on a Bruker AVANCE III-400. Chemical shifts are given on the δ
■
University, where a voucher specimen (accession number: 20100720)
was deposited.
Extraction and Isolation. The air-dried roots of I. racemosa (10.0
kg) were percolated with MeOH (3 × 20 L) at room temperature. The
combined extracts were concentrated under reduced pressure, and the
residue (3.2 kg) was partitioned into H O and extracted with
2
petroleum ether, EtOAc, and n-BuOH. The petroleum ether fraction
(600 g) was subjected to flash column chromatography (CC) over
silica gel (petroleum ether−EtOAc from 1:0 to 0:1, v/v) to give five
major fractions (A−E). Fraction A (179 g) was recrystallized from
petroleum ether to yield solid alantolides (153 g, a mixture of 6 and 8)
and a mother liquor (10 g). The mother liquor was subjected to CC
over silica gel (petroleum ether−EtOAc from 50:1 to 12:1, v/v) to
afford three fractions, A −A . Fraction A (3.2 g) was recrystallized
(
ppm) scale using TMS as internal standard. ESI-LRMS was
performed on a Finnigan LC QDECA instrument, and HRESIMS
was performed on a Bruker APEX-II mass spectrometer. The X-ray
diffraction data were collected on a Bruker Smart Apex CCD
diffractometer, and the structures were solved by direct methods using
Bruker SHELXS-97 (using graphite-monochromated Mo Kα
radiation). Silica gel (200−300 mesh, Qingdao Marine Chemical
Factory, China), Sephadex LH-20 (Amersham Pharmacia Biotech),
and YMC Gel (ODS-A, 12 nm, S-50 μm, YMC Co., Kyoto, Japan)
were used for column chromatography. Silica gel GF254 plates (10−
1
3
1
from petroleum ether and subjected to repeated CC over silica gel
(petroleum ether− EtOAc, 20:1, v/v) to afford compounds 6 (20 mg)
and 7 (12 mg). Fraction A (3.0 g) was subjected to CC over silica gel
2
4
0 μm, Qingdao Marine Chemical Factory, China) were used for TLC.
The spots on TLC were detected with 254 nm UV light and visualized
(petroleum ether−EtOAc, 12:1, v/v) to afford 10 (30 mg) and 11 (50
mg). Fraction A3 (2.7 g) was chromatographed over silica gel
(petroleum ether−EtOAc, 12:1, v/v) and repeated CC over Sephadex
by spraying with 98% H SO −C H OH (5:95, v/v) followed by
2
4
2
5
heating.
LH-20 (CHCl −MeOH, 2:1, v/v) to obtain compounds 8 (20 mg)
3
Plant Material. The roots of I. racemosa were collected from
Gannan, Gansun Province, China, in July 2010. The plant was
identified by Prof. Guo-Liang Zhang, School of Life Sciences, Lanzhou
and 9 (70 mg). Fraction B (1.5 g) was subjected to CC over silica gel
(petroleum ether−EtOAc, 8:1, v/v) to give fractions B and B2.
1
Fraction B (300 mg) was separated on a silica gel column (petroleum
1
E
dx.doi.org/10.1021/np300742d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ether−EtOAc, 5:1, v/v) to obtain 12 (60 mg) and 13 (15 mg).
C H O , space group P2(1), a = 6.155(8) Å, b = 11.287(15) Å, c =
15
22
4
Fraction B (750 mg) was purified by CC over silica gel (petroleum
9.836(13) Å, α = 90.00°, β = 96.098(14), γ = 90.00°, V = 679.5(16)
2
3
3
ether−EtOAc, 5:1, v/v) to give two major subfractions, B and B2‑2.
Å , Z = 2, D =1.302 g/cm , R1 = 0.0523, wR2 = 0.1282.
2
‑1
calc
Fraction B2 (450 mg) was applied to CC over ODS (MeOH−H O,
Crystal Data for 5. Racemosalactone D (5) was crystallized from
‑1
2
1
:1, v/v) to afford 15 (5 mg) and 14 (40 mg). Fraction B2‑2 (150 mg)
CHCl to give colorless crystals. A single crystal of dimensions 0.25 ×
3
was subjected to CC over silica gel (petroleum ether−EtOAc, 4:1, v/v)
to yield 1 (8 mg). Fraction C (2.1 g) was separated by CC over silica
3
0
.23 × 0.21 mm was used for X-ray measurements. Crystal data:
C H O , space group P2(1), a = 8.379(6) Å, b = 9.018(6) Å, c =
1
5
22
3
gel (petroleum ether−EtOAc, 5:1, v/v) to give two fractions, C and
3
1
1
9.545(14) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 1476.8(18) Å ,
Z = 4, Dcalc = 1.207 g/cm , R1 = 0.0455, wR2 = 0.0999.
C . Fraction C (900 mg) was purified by CC over silica gel
3
2
1
(
petroleum ether−EtOAc, 4:1, v/v) to give 16 (30 mg), 17 (15 mg),
The supplementary crystallographic data for 1, 2, 4, and 5 reported
in this paper have been deposited at the Cambridge Crystallographic
Data Centre, under the reference numbers CCDC 897955, 897960,
and 3 (20 mg). Fraction C (600 mg) was subjected to CC over silica
2
gel (petroleum ether−EtOAc, 3:1, v/v) to give 18 (30 mg). Fraction D
(
3 g) was purified by CC over silica gel (petroleum ether−EtOAc, 5:1,
8
97961, and 897956, respectively. Copies of the data can be obtained,
v/v) to give two major fractions, D and D . Fractions D (1.7 g) was
1
2
1
free of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, fax: +44 1223 336033 or e-mail: data_
request@ccdc.cam.ac.uk.
Cell Culture. Human non-small-cell lung cancer A549 cells were
cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine
separated on a silica gel column (petroleum ether−EtOAc, 3:1, v/v),
Sephadex LH-20 (CHCl −MeOH, 2:1, v/v), and then ODS (MeOH−
3
H O, 1:1, v/v) to yield 19 (50 mg), 20 (90 mg), and 5 (7 mg).
2
Fraction D (1.0 g) was applied to CC over ODS (MeOH−H O, 1:1,
2
2
v/v) to afford 21 (25 mg) and 22 (43 mg). Fraction E (3 g) was
separated on a silica gel column (petroleum ether−EtOAc, 2:1, v/v) to
give two fractions, E and E . Fraction E (500 mg) was purified by CC
serum (Gibco) and penicillium/streptomycin in 5% CO at 37 °C.
2
Hepatocellular carcinoma HepG2 cells were cultured in Dulbecco’s
modified essential medium (Gibco) with the same supplements as
above. Human fibrosarcoma HT1080 cells were cultured in Alpha
minimum essential medium with the same supplements as above.
HUVECs were purchased from PriCells (Wuhan, China) and
routinely cultured in endothelial cell medium (M&C Gene
Technology, China), supplemented with 0.03 mg/mL endothelial
cell growth supplement (M&C Gene Technology, China), 10% fetal
bovine serum, 0.1 mg/mL heparin (M&C Gene Technology, China),
and penicillium/streptomycin. HUVECs were used from the second to
the sixth passages for the experiments.
1
2
1
over ODS (MeOH−H O, 1:1, v/v) to afford crude 4 (5 mg) and 23
2
(
20 mg), and then they were recrystallized from MeOH, respectively.
Fraction E (1.0 g) was applied to CC over ODS (MeOH−H O, 2:3,
2
2
v/v) and then Sephadex LH-20 (CHCl −MeOH, 1:1, v/v) to afford 2
3
(
13 mg) and 24 (5 mg).
Racemosalactone A (1): colorless crystals (CHCl ); mp 125−126
3
°
C; [α]25 +100 (c 0.1, acetone); IR (KBr) ν 3439, 2926, 1733,
1
D
max
−1
1
13
644, 1266 cm ; H (400 MHz) and C NMR (100 MHz) data, see
Tables 1 and 2; HRESIMS m/z 233.1531 [M + H − H O] (calcd for
C H O , 233.1536).
+
2
1
5
21
2
Racemosalactone B (2): colorless, flaky crystals (MeOH); mp
25
In Vitro Matrigel Angiogenesis Assays. According to the
2
2
22−224 °C; [α] +30 (c 0.1, MeOH); IR (KBr) ν 3463, 3268,
max
−1 1 13
D
21
modified protocols, in vitro Matrigel angiogenesis assays were
928, 1748, 1645, 1224 cm ; H (400 MHz) and C NMR (100
conducted in 96-well plates using HUVEC cells plated on a Matrigel
layer (BD Biosciences) with 25 000 cells per well and incubated with
test compounds for 22 h. Images were taken 22 h after plating.
Colchicine was used as a positive control. Tube formation was
quantified by measuring the total length of capillary structures using
the software WCIF ImageJ. Two representative fields were counted in
each experiment. Magnification was ×40.
MHz) data, see Tables 1 and 2; ESIMS m/z 301, 302, 303, 318, 319,
+
3
20; HRESIMS m/z 318.1467 [M + NH ] (calcd for C H ClNO ,
4 15 25 4
3
18.1467).
Racemosalactone C (3): colorless oil; [α]25D −40 (c 0.1, acetone);
−1
1
IR (KBr) ν 3448, 2929, 1749, 1651, 1198, 983 cm ; H (400
max
MHz) and 13C NMR (100 MHz) data, see Tables 1 and 2; HRESIMS
+
m/z 251.1650 [M + H] (calcd for C H O , 251.1642).
15
23
3
Racemosalactone D (4): colorless crystals (CHCl ); mp 170−172
Cell Proliferation Assay. Cell proliferation was determined using
CCK-8 dye (Beyotime Inst Biotech, China) according to the
3
°
C; [α]25 +100 (c 0.1, acetone); IR (KBr) ν 3492, 3320, 2948,
1
D
max
−1 1
13
770, 1649, 1212 cm ; H (400 MHz) and C NMR (100 MHz)
22
manufacturer’s instructions. Briefly, cells were seeded in a 96-well
+
data, see Tables 1 and 2; HRESIMS m/z 284.1859 [M + NH ] (calcd
4
plate and allowed to adhere overnight. After incubation with
for C H NO , 284.1856).
15
26
4
compounds under evaluation for 72 h at 37 °C in 5% CO , 10 μL
2
Racemosalactone E (5): colorless crystals (CHCl ); mp 80−81 °C;
3
of CCK-8 dyes then was added to each well, and cells were incubated
25
[
α] −140 (c 0.1, acetone); IR (KBr) ν 3511, 2959, 1742, 1640,
D
max
at 37 °C in 5% CO for 2 h. Mitomycin was used as a positive control.
−1 1 13
2
1
1
2
210 cm ; H (400 MHz) and C NMR (100 MHz) data, see Tables
and 2; HRESIMS m/z 268.1912 [M + NH ] (calcd for C H NO ,
+
All experiments were performed in duplicate. The absorbance was
determined at 450 nm using a microplate reader (TECAN Infinite
M200).
Oxidation of Alantolides. A suspension of selenium dioxide (296
mg, 2.64 mmol) in DCM (10 mL) at 0 °C under Ar was added to 640
μL (5.72 mmol) of a 70% solution of tert-butyl hydroperoxide. After
stirring for 30 min, a solution of alantolides (1.20 g) in 10 mL of DCM
was added dropwise. The mixture was stirred at room temperature for
4
15 26
3
68.1907).
X-ray Analysis. All measurements were collected on a Bruker
Smart Apex CCD diffractometer with graphite-monochromated Mo
Kα radiation (λ = 0.71073 Å). The structures of 1, 2, 4, and 5 were
solved by direct methods (SHELX97).
Crystal Data for 1. Racemosalactone A (1) was crystallized from
CHCl to give colorless crystals. A single crystal of dimensions 0.21 ×
3
3
0
.20 × 0.19 mm was used for X-ray measurements. Crystal data:
4 h. Aqueous NaHCO was added, and the mixture extracted with
3
C H O , space group P2(1), a = 12.070(10) Å, b = 7.880(6) Å, c =
DCM, washed with brine, and dried over Na SO . The residue was
1
5
22
3
2
4
1
4.734(12) Å, α = 90.00°, β = 93.781(9)°, γ = 90.00°, V = 1398.4(19)
purified by column chromatography on silica gel (eluent, petroleum
ether−EtOAc, 3:1) to give a crude solid. Recrystallization from
petroleum afforded 6 (0.60 g) and 17 (0.40 g, 90%).
3
3
Å , Z = 4, D = 1.189 g/cm , R1 = 0.0489, wR2 = 0.1068.
calc
Crystal Data for 2. Racemosalactone B (2) was crystallized from
MeOH to give colorless, flaky crystals. A single crystal of dimensions
0
data: C H ClO , space group P2(1), a = 6.422(4) Å, b = 11.870(7)
Å, c = 9.689(6) Å, α = 90.00°, β = 103.415(6)°, γ = 90.00°, V =
3
.20 × 0.16 × 0.14 mm was used for X-ray measurements. Crystal
ASSOCIATED CONTENT
15
21
4
■
*
S
Supporting Information
3
3
7
18.4(7) Å , Z = 2, Dcalc = 1.390 g/cm , R1 = 0.0397, wR2 = 0.0760.
Crystal Data for 4. Racemosalactone D (4) was crystallized from
1
5
D and 2D NMR, IR, and HRESIMS spectra of compounds 1−
and crystal data of compounds 1, 2, 4, and 5. This material is
CHCl to give colorless crystals. A single crystal of dimensions 0.23 ×
0
3
3
.22 × 0.19 mm was used for X-ray measurements. Crystal data:
available free of charge via the Internet at http://pubs.acs.org.
F
dx.doi.org/10.1021/np300742d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
AUTHOR INFORMATION
Corresponding Author
Tel: +86-931-8912592. Fax: +86-931-8912582. E-mail:
npchem@lzu.edu.cn.
■
*
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (No. 31270396) and the 111 Project of
China.
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dx.doi.org/10.1021/np300742d | J. Nat. Prod. XXXX, XXX, XXX−XXX