Guaiane sesquiterpene lactones and amino acid-sesquiterpene lactone conjugates from the aerial parts of Saussurea pulchella

Article abstract of DOI:10.1021/np800005r

Two new guaiane sesquiterpene lactones (1 and 2) and seven new amino acid-sesquiterpene lactone conjugates (3-9), together with six known sesquiterpene lactones (10-15), were isolated from the methanol extract of the aerial parts of Saussurea pulchella. Their structures were determined on the basis of spectroscopic and chemical methods to be 8α-O-(3′-hydroxy- 3′-methylbutyryl)desacylcynaropicrin (1), 8α-O-(2′, 3′-dihydroxyisobutyryl)l 11β,13-dihydrodesacylcynaropicrin (2), and pulchellamines A, B, C, D, E, F, and G (3-9). The structures of the new amino acid-sesquiterpene lactone conjugates, pulchellamines A, B, C, D, E, F, and G (3-9), were confirmed by synthesis. The isolated compounds were evaluated for cytotoxic activity against four human tumor cell lines. Compounds 11 and 12 exhibited cytotoxicity against skin melanoma (SK-MEL-2) and ovary malignant ascites (SK-OV-3) human tumor cell lines with ED₅₀ values of 1.53 and 4.07 μM, and 2.49 and 7.42 μM, respectively.

Full text of DOI:10.1021/np800005r

678
J. Nat. Prod. 2008, 71, 678–683
Guaiane Sesquiterpene Lactones and Amino Acid-Sesquiterpene Lactone Conjugates from the
Aerial Parts of Saussurea pulchella
Min Cheol Yang,† Sang Un Choi,‡ Wahn Soo Choi,§ Sun Yeou Kim, and Kang Ro Lee*,†
Natural Products Laboratory, College of Pharmacy, Sungkyunkwan UniVersity, Suwon 440-746, Korea, Korea Research Institute of Chemical
Technology, Teajeon 305-600, Korea, Department of Immunology, College of Medicine, Konguk UniVersity, Chungju 380-704, Korea, and
Department of Herbal Pharmacology, Graduate School of East-West Science, Kyung Hee UniVersity, Suwon 446-701, Korea
ReceiVed January 3, 2008
Two new guaiane sesquiterpene lactones (1 and 2) and seven new amino acid-sesquiterpene lactone conjugates (3-9),
together with six known sesquiterpene lactones (10-15), were isolated from the methanol extract of the aerial parts of
Saussurea pulchella. Their structures were determined on the basis of spectroscopic and chemical methods to be 8R-
O-(3′-hydroxy-3′-methylbutyryl)desacylcynaropicrin (1), 8R-O-(2′, 3′-dihydroxyisobutyryl)11ꢀ,13-dihydrodesacylcy-
naropicrin (2), and pulchellamines A, B, C, D, E, F, and G (3-9). The structures of the new amino acid-sesquiterpene
lactone conjugates, pulchellamines A, B, C, D, E, F, and G (3-9), were confirmed by synthesis. The isolated compounds
were evaluated for cytotoxic activity against four human tumor cell lines. Compounds 11 and 12 exhibited cytotoxicity
against skin melanoma (SK-MEL-2) and ovary malignant ascites (SK-OV-3) human tumor cell lines with ED50 values
of 1.53 and 4.07 µM, and 2.49 and 7.42 µM, respectively.
In a continuing search for bioactive constituents from Korean
Asteraceae medicinal plants,1,2 the sesquiterpene lactones from the
aerial parts of Saussurea pulchella Fisch (Asteraceae) were
investigated. Sesquiterpenes,3–7 lignans,8–10and flavonoids11,12 have
been reported as constituents of the genus Saussurea and have been
found to possess a wide range of biological activities, including
cytotoxic,3–5 anti-inflammation,6,7 and antioxidant activities.11,12
Although there have been a number of studies on the chemical
constituents and biological activities of the genus Saussurea, there
have been few phytochemical investigations on S. pulchella, and
to date only a sesquiterpene lactone13,14 and four phenolics15,16
have been reported. S. pulchella is widely distributed in Korea and
has been used in Korean traditional medicine for the treatment of
inflammation, hypertension, hepatitis, and arthritis.17 We reported
lignan and terpene constituents from the MeOH extract of the aerial
parts of S. pulchella.18 In our continuing study on this plant source,
we have identified two new guaiane sesquiterpene lactones (1 and
2) and seven new amino acid-sesquiterpene lactone conjugates
(3-9), together with six known sesquiterpene lactones (10-15),
from the MeOH extract. The structures were determined on the
basis of spectroscopic and chemical methods. Here we report the
isolation, structural characterization, and cytotoxicity of the isolated
sesquiterpene lactones.
the molecular ion peak at m/z 362.1732 [M]+ (calcd for C20H26O6,
362.4228) obtained by HREIMS. The IR spectrum displayed
absorption bands for hydroxy (3401 cm-1), γ-lactone (1766 cm-1),
1
and ester functional groups (1732 cm-1). The H and 13C NMR
chemical shifts of H-13 at δ 6.25 (d, J ) 3.5 Hz) and 5.66 (d, J )
3.0 Hz), H-6 at δ 4.23 (dd, J ) 10.0, 9.0 Hz), H-7 at δ 3.12 (dddd,
J ) 10.0, 9.0, 3.5, 3.0 Hz), and C-12 at δ 168.9 indicated the
presence of an R-exomethylene-6,7-γ-lactone moiety. Additionally,
1
the H and 13C NMR spectra of 1 displayed resonances for two
exomethylenes {δ 5.16 (s), 4.97 (d, J ) 1.0 Hz); δ 141.6, 118.2,
5.50 (t, J ) 2.0 Hz), 5.37 (t, J ) 1.5 Hz); δ 152.2, 113.6}, two
oxygenated methines {δ 4.57 (dddd, J ) 7.5, 7.0, 2.0, 1.5 Hz); δ
73.7, 5.09 (m); δ 74.2}, and two methines {δ 2.98 (ddd, J ) 10.0,
9.0, 7.5 Hz); δ 45.3, 2.84 (dd, J ) 10.5, 9.0 Hz); δ 51.3}. These
data were similar to those of 10, which was also isolated from this
plant source. The difference was only the addition of a 3-hydroxy-
3-methylbutyryl group {δ 2.59 (2H, s, H-2′); δ 46.5 (C-2′), 1.36
(6H, d, J ) 3.0 Hz, H-4′, 5′); δ 29.3 (C-4′), 29.2 (C-5′), 172.0
(C-1′), and 69.2 (C-3′)}.27 In the HMBC spectrum, correlation of
H-8 (δ 5.09, m) with C-1′ (δ 172.0) supported the connectivity of
a 3-hydroxy-3-methylbutyrate group at C-8 (Figure 2). According
to the J values in the 1H NMR spectrum, the stereochemistry of 1
was expected to be the same as 10. This was confirmed by the
NOESY correlations (Figure 2). On the basis of these findings, the
structure of 1 was determined to be 8R-O-(3′-hydroxy-3′-methyl-
butyryl)desacylcynaropicrin.
Results and Discussion
Column chromatographic separation of the MeOH extract of S.
pulchella afforded two new guaiane sesquiterpene lactones (1 and
2) and seven new amino acid-sesquiterpene lactone conjugates
(3-9), as well as six known compounds, desacylcynaropicrin
(10),19,20 8R-(4′-hydroxysenecioyloxy)dehydrozaluzanin C (11),21
cynaropicrin (12),22 11ꢀ,13-dihydrodesacylcynaropicrin (13),23,24
3R-dihydro-4(15)-dehydrogrosshemin R,ꢀ-dihydroxyisoburyrate
(14),25 and 11ꢀ,13-dihydrodesacylcynaropicrin 8-ꢀ-D-glucoside
(15).26 (Figure 1).
Compound 2 was obtained as a colorless oil, [R]D +24.1 (c 0.1,
MeOH). The molecular formula of C19H26O7 was determined from
the molecular ion peak at m/z 366.1679 [M]+ (calcd for C19H26O7,
366.4112) in the HREIMS. The IR spectrum showed a γ-lactone
(1769 cm-1) and an ester functional group (1735 cm-1). The IR
1
and H and 13C NMR data of 2 were very similar to those of 13,
which was isolated from this plant. The differences were due to
the addition of an R,ꢀ-dihydroxyisobutyryl group {δ 3.61 (1H, d,
J ) 10.5 Hz, H-3′), 3.75 (1H, d, J ) 10.5 Hz, H-3′); δ 69.3 (C-3′),
1.32 (3H, s, H-4′); δ 22.3 (C-4′), 175.6 (C-1′), and 76.9 (C-2′)}.25
In the HMBC spectrum, correlation of the H-8 (δ 5.02, m) with
C-1′ (δ 175.6) supported the connectivity of the R,ꢀ-dihydroxy-
Compound 1 was obtained as a colorless oil, [R]D +23.0 (c 0.3,
MeOH). The molecular formula of C20H26O6 was determined from
* Corresponding author. E-mail: krlee@skku.ac.kr. Tel: 82-31-290-7710.
Fax: 82-31-292-8800.
1
isoburyrate at C-8. According to the H and 13C NMR data, the
† Sungkyunkwan University.
relative configuration of 2 is expected to be the same as 13. This
was confirmed from the correlations observed in the NOESY
spectrum. The configuration at C-2′ was not determined, however.
‡ Korea Research Institute of Chemical Technology.
§ Konguk University.
Kyung Hee University.
10.1021/np800005r CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/04/2008

Guaiane Sesquiterpene Lactones from Saussurea pulchella
Journal of Natural Products, 2008, Vol. 71, No. 4 679
Figure 1. Structures of compounds 1-9 isolated from the MeOH extract of S. pulchella.
Figure 3. Key HMBC (f) and NOESY(T) correlations of 3.
Figure 2. Key HMBC (f) and NOESY(T) correlations of 1.
J ) 17.1, 6.1 Hz, H-3′′b); 13C NMR δ 174.5 (C-1′′), 59.3 (C-2′′),
33.9 (C-3′′), 172.8 (C-4′′)},6,28 and the replacement of a CH3 group
{δ 1.42 (d, J ) 7.0 Hz), 15.9} with a methylene moiety {δ 3.64
(dd, J ) 12.6, 5.2 Hz), 3.42 (dd, J ) 12.6, 8.1 Hz), 47.3}. The
resonances of H-13a (δ 3.64, dd, J ) 12.6, 5.2 Hz) and H-13b (δ
3.42, dd, J ) 12.6, 8.1 Hz) showed correlations with the methine
carbon (δ 59.3, C-2′′) of the L-asparagine moiety in the HMBC
spectrum (Figure 3), indicating the position of the L-asparagine
moiety at C-13. On the basis of the J values of the 1H NMR data,
the stereochemistry of 3 was proposed to be the same as 13. This
was confirmed by the NOESY results (Figure 3). Thus, the structure
of 3 was determined and it was named pulchellamine A. The
structure of pulchellamine A was confirmed by synthesis from
Thus, the structure of 2 was determined to be 8R-O-(2′, 3′-
dihydroxyisobutyryl)11ꢀ,13-dihydrodesacylcynaropicrin.
Compound 3, a colorless gum, [R]D +41.4 (c 0.3, MeOH), was
deduced to possess a nitrogen function on the basis of the ninhydrin
test. The molecular formula of C19H27N2O7 was determined from
the molecular ion peak at m/z 395.1827 [M + H]+ (calcd for
C19H27N2O7H, 395.1812) obtained by HRESIMS. The IR spectrum
exhibited absorption bands for hydroxy (3338 cm-1) and γ-lactone
1
functional groups (1762 cm-1). The H and 13C NMR data were
very similar to those of 13, except for the additional appearance of
the side chain moiety, an L-asparagine {1H NMR δ 3.97 (dd, J )
6.1, 4.5 Hz, H-2′′), 3.02 (dd, J ) 17.1, 4.5 Hz, H-3′′a), 2.96 (dd,

680 Journal of Natural Products, 2008, Vol. 71, No. 4
Yang et al.
desacylcynaropicrin (10) and L-asparagine using a Michael-type
addition reaction.6 The synthesized 3 was identified by comparison
of its spectroscopic data (1H NMR, ESIMS, and [R]D) with those
of the isolated compound.
confirmed by the HMBC spectrum. The H-13a (δ 3.98, m) and
H-13b (δ 3.12, br t, J ) 11.6 Hz) resonances showed correlations
with the methine carbon (δ 64.3, C-2′′) of the L-phenylalanine
moiety. The stereochemistry of 6 was assumed to be the same as
4, on the basis of the J values. This was confirmed by a NOESY
experiment. Thus, the structure of 6 was determined and it was
named pulchellamine D. Pulchellamine D was synthesized from
desacylcynaropicrin (10) and L-phenylalanine.6 The synthesized
pulchellamine D was identified by comparison of its [R]D, 1H NMR,
and ESIMS data with those of the isolated compound (6).
Compound 4, a white crystal from MeOH-H2O, mp 198 °C,
[R]D +52.0 (c 0.15, MeOH), showed a positive ninhydrin test. The
molecular formula of C19H27NO6 was determined from the molec-
ular ion peak at m/z 366.1927 [M + H]+ (calcd for C19H27NO6H,
366.1911) obtained by HRESIMS. Its IR spectrum revealed
absorption bands for hydroxy (3297 cm-1) and γ-lactone functional
1
groups (1764 cm-1). The H and 13C NMR data were similar to
Compound 7, a white crystal from MeOH-H2O, mp 220 °C,
[R]D +29.0 (c 0.2, MeOH), was deduced to possess a nitrogen
function on the basis of the ninhydrin test. The molecular formula
of C20H29NO6 was determined from the molecular ion peak at m/z
380.2067 [M + H]+ (calcd for C20H29NO6H, 380.2068) obtained
by HRESIMS. The IR spectrum revealed absorption bands for
hydroxy (3333 cm-1) and γ-lactone functional groups (1765 cm-1).
The 1H and 13C NMR data were similar to those of 6. The 1H and
13C NMR spectra showed the characteristics of an L-valine moiety
{1H NMR δ 2.82 (m, H-2′′), 1.88 (m, H-3′′), 0.87 (3H, d, J ) 6.6
Hz, H-4′′), 0.90 (3H, d, J ) 6.6 Hz, H-5′′); 13C NMR δ 173.6
(C-1′′), 67.6 (C-2′′), 22.0 (C-3′′), 19.2 (C-4”), 18.3 (C-5′′)}.6,28 The
H-13a (δ 3.18, m) and H-13b (δ 2.87, m) resonances showed
HMBC correlations with the methine carbon (δ 67.6, C-2′′) of the
L-valine moiety. The stereochemistry of 7 was assumed to be the
same as 13, on the basis of the J values of the 1H NMR data. This
was confirmed from the correlations observed in the NOESY
spectrum. Thus, the structure of 7 was determined and it was named
pulchellamine E. The structure of pulchellamine E was confirmed
by synthesis from desacylcynaropicrin (10) and L-valine using a
Michael-type addition reaction.6 The synthesized 7 was identified
those of 3, except for a 4-aminobutanoic acid moiety {1H NMR δ
2.45 (td, J ) 11.4, 7.2 Hz, H-2′′a), 2.60 (m, H-2′′b), 1.62 (q, J )
7.2 Hz, H-3′′), 2.22 (2H, t, J ) 7.2 Hz, H-4′′); 13C NMR δ 174.4
(C-1′′), 47.9 (C-2′′), 24.3 (C-3′′), 31.9 (C-4′′)}.29,30 The H-13a
resonances (δ 3.01, dd, J ) 12.6, 3.0 Hz) and H-13b (δ 2.53, dd,
J ) 12.6, 4.8 Hz) showed correlations with the methylene carbon
(δ 31.9, C-4′′) of the 4-aminobutanoic acid moiety in the HMBC
spectrum, indicating the position of this moiety at C-13. The
stereochemistry of 4 was proposed to be the same as 3, on the
basis of the J values. This was confirmed by a NOESY experiment.
Thus, the structure of 4 was determined and it was named
pulchellamine B. The structure of pulchellamine B was confirmed
by synthesis from desacylcynaropicrin (10) and 4-aminobutanoic
acid using a Michael-type addition reaction.6 The synthesized 4
was identified by comparison of its spectroscopic data (1H NMR,
ESIMS, and [R]D) with those of the isolated compound.
Compound 5, a colorless gum, [R]D +22.0 (c 0.5, MeOH),
showed a positive ninhydrin test. The molecular formula of
C24H33NO9was determined from the molecular ion peak at m/z
480.2249 [M + H]+ (calcd for C24H33NO9H, 480.2228) obtained
by HRESIMS. The IR spectrum displayed absorption bands for
hydroxy (3305 cm-1) and γ-lactone functional groups (1755 cm-1).
1
by comparison of its [R]D, H NMR, and ESIMS data with those
of the isolated compound.
1
The H and 13C NMR data were similar to those of 2, except for
Compound 8, a colorless gum, [R]D +40.4 (c 0.4, MeOH),
showed a positive ninhydrin test. The molecular formula of
C26H30N2O6 was determined from the molecular ion peak at m/z
467.2190 [M + H]+ (calcd for C26H30N2O6H, 467.2176), obtained
by HRESIMS. The IR spectrum showed absorption bands for
hydroxy (3301 cm-1), γ-lactone (1762 cm-1), and CdC functional
the appearance of an L-proline moiety {1H NMR δ 3.79 (t, J ) 7.0
Hz, H-2′′), 2.21 (2H, m, H-3′′), 1.67, 1.84 (each m, H-4′′a, 4′′b),
3.46 (dt, J ) 10.0, 5.0 Hz, H-5′′a), 2.76 (q, J ) 10.0 Hz, H-5′′b);
13C NMR δ 177.2 (C-1′′), 68.8 (C-2′′), 30.5 (C-3′′), 25.1 (C-4′′),
55.5 (C-5′′)}6,28 and the replacement of a CH3 group {δ 1.42 (d, J
) 7.0 Hz), 15.9} with methylene {δ 3.67 (dd, J ) 13.5, 6.0 Hz),
2.53 (d, J ) 13.5 Hz), 55.3}. The H-13a (δ 3.67, dd, J ) 13.5, 6.0
Hz) and H-13b (δ 3.56, d, J ) 13.5 Hz) resonances showed
correlations with the methine carbon (δ 68.8, C-2′′) of the L-proline
moiety in the HMBC spectrum, indicating the position of the
L-proline moiety at C-13. The stereochemistry of 5 was propsed to
1
groups (1634 cm-1). The H and 13C NMR data were similar to
those of 6 and 7. The differences were the amino acid (L-tryptophan)
moiety {1H NMR δ 3.87 (t, J ) 5.5 Hz, H-2′′), 3.41, 3.87 (each
m, H-3′′a, 3′′b), 7.39 (d, J ) 8.0 Hz, H-6′′), 7.07 (dt, J ) 7.0, 1.0
Hz, H-7′′), 7.14 (dt, J ) 7.0, 1.0 Hz, H-8′′), 7.69 (d, J ) 8.0 Hz,
H-9′′), 7.24 (s, H-11′′); 13C NMR δ 173.6 (C-1′′), 63.7 (C-2′′), 26.4
(C-3′′), 108.3 (C-4′′), 128.8 (C-5′′), 122.9 (C-6′′), 120.4 (C-7′′),
119.5 (C-8′′), 112.9 (C-9′′), 138.3 (C-10′′), 125.8 (C-11′′)}.6,28 The
H-13a (δ 3.87, m) and H-13b (δ 2.99, dd, J ) 12.5, 10.0 Hz)
resonances showed HMBC correlations with the methine carbon
(δ 63.7, C-2′′) of the L-tryptophan moiety. The stereochemistry of
8 was assumed to be the same as 7, on the basis of the J values of
1
be the same as 2, based on the J value of the H NMR data. This
was confirmed by a ROESY experiment. The configuration at C-2′
was not determined. Thus, the structure of 5 was determined and
it was named pulchellamine C. The structure of pulchellamine C
was confirmed by synthesis from 3R-dihydro-4(15)-dehydro-
grosshemin R,ꢀ-dihydroxyisoburyrate (14) and L-proline using a
Michael-type addition reaction.6 The synthesized 5 was identified
by comparison of its 1H NMR, ESIMS, and [R]D value with those
of the isolated compound.
1
the H NMR data and the data of the NOESY experiment. Thus,
the structure of 8 was determined and it was named pulchellamine
F. The structure of pulchellamine F was confirmed by synthesis
from desacylcynaropicrin (10) and L-tryptophan using a Michael-
type addition reaction.6 The 1H NMR, ESIMS, and [R]D values of
the synthetic 8 was identical to the data obtained for the isolated
pulchellamine F.
Compound 6, a colorless gum, [R]D +74.0 (c 0.3, MeOH),
showed a positive ninhydrin test. The molecular formula of
C24H29NO6 was determined from the molecular ion peak at m/z
428.2080 [M + H]+ (calcd for C24H29NO6H, 428.2067) obtained
by HRESIMS. The IR spectrum displayed absorption bands for
Compound 9, a white crystal from MeOH-H2O, mp 199 °C,
[R]D +15.4 (c 0.1, MeOH), showed a positive ninhydrin test. The
molecular formula of C21H31NO6was determined from the molecular
ion peak at m/z 394.2214 [M + H]+ (calcd for C21H31NO6H,
394.2224), obtained by HRESIMS. The IR spectrum displayed
absorption bands for hydroxy (3372 cm-1) and γ-lactone functional
1
hydroxy (3333 cm-1) and γ-lactone groups (1763 cm-1). The H
and 13C NMR data were similar to those of 4. The spectra showed
the characteristic signals of an L-phenylalanine moiety {1H NMR
δ 3.98 (m, H-2′′), 3.33 (2H, d, J ) 6.1 Hz, H-3′′), 7.29 (2H, t, J )
7.3 Hz, H-5′′, 9′′), 7.49 (2H, d, J ) 7.3 Hz, H-6′′, 8′′), 7.22 (t, J )
8.0 Hz, H-7′′); 13C NMR δ 176.4 (C-1′′), 64.3 (C-2′′), 40.2 (C-
3′′), 138.8 (C-4′′), 130.5 (C-5′′, 9′′), 129.1 (C-6′′, 8′′), 127.3
(C-7′′)}.6,28 The position of the L-phenylalanine moiety was
1
groups (1766 cm-1). The H NMR data of the sesquiterpene part
in 9 is rather different from those of 7 and 8. The differences were
the amino acid (L-isoleucine) moiety {1H NMR δ 2.96 (d, J ) 5.0

Guaiane Sesquiterpene Lactones from Saussurea pulchella
Journal of Natural Products, 2008, Vol. 71, No. 4 681
Table 1. 1H NMR Data of Compounds 1-4
1a
2b
3c
4d
H-1
2.98 (ddd, 10.5, 9.0, 7.5)
2.99 (ddd, 10.0, 9.0, 7.5)
2.20 (ddd, 13.0, 7.5, 7.0)
1.71 (ddd, 13.0, 10.0, 8.5)
4.52 (dddd, 8.5, 7.0, 2.5, 2.0) 4.62 (br t, 5.5)
2.85 (m)
3.04 (m)
2.36 (td, 13.1, 5.5)
1.71 (td, 13.1, 8.5)
2.80 (m)
2.07 (m)
1.52 (td, 12.6, 8.4)
4.31 (br t, 8.4)
H-2a 2.22 (ddd, 13.5, 7.5, 7.0,)
H-2b 1.71 (ddd, 13.5, 10.5, 7.5,)
H-3
H-5
H-6
H-7
H-8
4.57 (dddd, 7.5, 7.0, 2.0, 1.5)
2.84 (dd, 10.5, 9.0)
4.23 (dd, 10.0, 9.0)
3.12 (dddd, 10.0, 9.0, 3.5, 3.0) 2.00 (ddd, 10.0, 10.0, 10.0)
5.09 (m)
3.04 (m)
4.36 (br t, 10.0)
2.49 (ddd, 10.0, 10.0, 10.0)
3.89 (m)
2.80 (m)
4.25 (t, 10.0)
4.01 (br t, 10.0)
2.12 (ddd, 10.0, 10.0, 10.0)
3.50 (m)
5.02 (m)
H-9a 2.70 (dd, 14.5, 5.5)
H-9b 2.37 (dd, 14.5, 4.0)
H-11
H-13 6.25 (d, 3.5)/5.66 (d, 3.0)
H-14 5.16 (s)/4.97 (d, 1.0)
H-15 5.50 (t, 2.0)/5.37 (t, 1.5)
H-2′ 2.59 (s, 2H)
H-3′
2.77 (dd, 13.5, 5.0)
2.22 (dd, 13.5, 6.0)
2.55 (dd, 10.0, 7.0)
1.42 (d, 7.0, 3H)
5.15 (s)/5.02 (s)
5.43 (d, 2.5)/5.30 (d, 2.0)
2.78 (dd, 13.0, 4.9)
2.27 (dd, 13.0, 7.9)
3.29 (m)
2.56 (m)
2.04 (dd, 12.6, 7.8)
2.71 (ddd, 10.0, 4.8, 3.0)
3.64 (dd, 12.6, 5.2)/3.42 (dd, 12.6, 8.1) 3.01 (dd, 12.6, 3.0)/2.53 (dd, 12.6, 4.8)
5.09 (2H, s)
5.33 (s)/5.30 (s)
4.92 (s)/4.87 (s)
5.14 (s)/5.10 (s)
3.61 (d, 10.5)/3.75 (d, 10.5)
1.32 (s, 3H)
H-4′ 1.36 (d, 3.0, 3H)
H-5′ 1.36 (d, 3.0, 3H)
H-2′′
3.97 (dd, 6.1, 4.5)
2.45 (td, 11.4, 7.2)/2.60 (m)
H-3′′
H-4′′
3.02 (dd, 17.1, 4.5)/2.96 (dd, 17.1, 6.1) 1.62 (2H, q, 7.2)
2.22 (2H, t, 7.2)
a Measured in CDCl3 at 500 MHz. b Measured in CD3OD at 500 MHz. c Measured in D2O at 500 MHz. d Measured in DMSO-d6 at 500 MHz.
240 × 10 mm, 40–63 µm, Merck Co., Germany) with a FMI QSY-0
Hz, H-2′′), 1.64 (m, H-3′′), 1.14, 1.47 (m, H-4′′a, 4′′b), 0.86 (3H,
d, J ) 7.0 Hz, H-5′′), 0.83 (3H, d, J ) 7.0 Hz, H-6′′); 13C NMR
(Fluid Metering Inc.) pump. Open column chromatography was
performed using Si gel (particle size 70–230 mesh and 230–400 mesh,
δ 173.2 (C-1′′), 66.4 (C-2′′), 36.9 (C-3′′), 24.9 (C-4′′), 11.5 (C-
5′′), 15.5 (C-6′′)}.6,28 The H-13a (δ 3.17, dd, J ) 12.0, 3.5 Hz)
Merck Co., Germany), Si gel 60 RP-18 (40–63 µm, Merck Co.,
Germany), and Sephadex LH-20 (Pharmacia Co., Sweden). Thin-layer
and H-13b (δ 2.55, m) resonances showed HMBC correlations with
the methine carbon (δ 66.4, C-2′′) of the L-isoleucine moiety. The
chromatography (TLC) was performed on Si gel 60 F254 and RP-18
F254s (Merck Co., Germany).
stereochemistry of 9 was assumed to be the same as 8, on the basis
Plant Material. S. pulchella was collected at Mt. Odae, Korea, in
1
of the J values of the H NMR data. This was confirmed by a
August 2005. A voucher specimen (SKK-05-080) was deposited at the
NOESY experiment. Thus, the structure of 9 was determined and
it was named pulchellamine G. The structure of pulchellamine G
was confirmed by synthesis from deacylcynaropicrin (10) and
L-isoleucine using a Michael-type addition reaction.6 The synthe-
sized 9 was identified by comparison of its [R]D, H NMR, and
ESIMS data with those of the isolated compound.
The isolated compounds were evaluated for cytotoxic activity
against four human tumor cell lines (Table 4). Compounds 11 and
12 exhibited cytotoxicity against skin melanoma (SK-MEL-2) and
ovary malignant ascites (SK-OV-3) human tumor cell lines with
ED50 values of 1.53 and 4.07 µM, and 2.49 and 7.42 µM,
respectively. Compounds 1, 5, and 10 were moderately cytotoxic
against a skin melanoma (SK-MEL-2) human tumor cell line with
ED50 values 6.78, 5.73, and 9.74 µM, respectively. The other
compounds showed little cytotoxicity against the tested cell lines.
Considering structure–activity relationships for the cytotoxicity
against four tested cancer cell lines of the isolated compounds, the
presence of a side chain at C-8 increases the cytotoxicity and the
R-exomethylene-γ-lactone ring is essential for cytotoxic activity.
College of Pharmacy at Sungkyunkwan University.
Extraction and Isolation. The partially dried and chopped aerial
parts of S. pulchella (3.5 kg) were extracted three times with MeOH at
room temperature. The resulting MeOH extract (350 g) was subjected
to successive solvent partition, giving n-hexane (70 g), CHCl3 (33 g),
EtOAc (21 g), and n-BuOH (40 g) soluble fractions. The CHCl3 fraction
(33 g) was chromatographed over a Si gel column using a gradient
solvent system of CHCl3-MeOH, 20:1–1:1, to give seven fractions
(C1-C7). Fraction C3 (2.2 g) was subjected to Sephadex LH-20
(CH2Cl2-MeOH, 1:1) and a Lichroprep Si 60 column (CHCl3-MeOH,
8:1) and purified using preparative HPLC (25% CH3CN) to afford 10
(90 mg, 0.0026% w/w), 11 (22 mg, 0.00063% w/w), and 1 (12 mg,
0.00034% w/w). Fraciton C4 (5.0 g) was subjected to Sephadex LH-
20 (CH2Cl2-MeOH, 1:1) and a Si gel column (CHCl3-EtOAc-MeOH,
8:8:1) and purified using a Lichroprep RP-18 column (35% CH3CN)
to afford 12 (33 mg, 0.00094% w/w). Fraction C5 (3.0 g) was subjected
to Sephadex LH-20 (CH2Cl2-MeOH, 1:1) and purified using a
Lichroprep RP-18 column (30% CH3CN) to afford 13 (68 mg, 0.0019%
w/w). Fraction C6 (4.5 g) was subjected to a Si gel column
(CH2Cl2-EtOAc-MeOH, 8:7:1) and Sephadex LH-20 (CH2Cl2-
MeOH, 1:1) and purified using a Lichroprep RP-18 column (25%
CH3CN) to afford 14 (115 mg, 0.0033% w/w) and 2 (18 mg, 0.00051%
w/w). The EtOAc fraction (21 g) was chromatographed over a Si gel
column using a gradient solvent system of CHCl3-MeOH, 12:1–7:1,
to give seven fractions (E1-E7). Fraction E6 (3.0 g) was subjected to
Sephadex LH-20 (100% MeOH) and a Lichroprep RP-18 column (20%
MeOH) and purified using preparative HPLC (20% MeOH) to afford
15 (118 mg, 0.0034% w/w).
The n-BuOH fraction (40 g) was chromatographed over a DIAION
HP-20 resin column using a gradient solvent system of 100% distilled
H2O and 100% MeOH to give four fractions (B1-B4). Fraction B2
(22.0 g) was subjected to RP-18 column chromatography (40% MeOH)
to give three subfractions (B21-B23). Subfraction B22 (13.0 g) was
subjected to RP-18 column chromatography (15% CH3CN) to give three
subfractions (B221-B225). Subfraction B221 (3.5 g) was subjected
to Sephadex LH-20 (70% MeOH) and a Si gel column (EtOAc-MeOH-
H2O, 9:3:1) and purified using preparative HPLC (EtOAc-
MeOH-H2O, 9:3:1) to afford 3 (15 mg, 0.00043% w/w). Subfraction
B222 (2.0 g) was subjected to Sephadex LH-20 (70% MeOH) and a
Si gel column (EtOAc-MeOH-H2O, 9:3:1) and purified by Lichroprep
1
Experimental Section
General Experimental Procedures. Melting points were determined
on a Gallenkamp melting point apparatus and were uncorrected. Optical
rotations were measured using a JASCO P-1020 polarimeter (JASCO
Co., Japan). UV spectra were recorded on an Agilent 8453 UV
spectrophotometer (Agilent Co.), and the IR spectra were recorded on
a Bruker IFS-66/S instrument (Bruker Co., Germany). NMR spectra
were obtained on either a Bruker Biospin Avance 500 (Bruker Co.,
Germany) or a Varian Unity INOVA 500 NB NMR spectrometer
(Varian Co.). The EI, FAB, ESIMS, HREIMS, and HRESIMS data
were obtained on a JEOL JMS 700 (JEOL Co., Japan) and Mariner
(Perseptive Biosysystem Co.) mass spectrometer. A Gilson preparative
HPLC (Gilson Co., France) with a refractive index detector (Shodex
RI-101, Shodex Co., Japan) and Econosil C18 column (10 × 250 mm,
10 µm, Alltech Co.) was used for preparative HPLC. Low-pressure
liquid chromatography was carried out using a Lobar-A glass prepacked
column (Lichroprep Si 60, 240 × 10 mm, 40–63 µm, Merck Co.,
Germany) and a Lobar-A glass prepacked column (Lichroprep RP-18,

682 Journal of Natural Products, 2008, Vol. 71, No. 4
Table 2. 1H NMR Data of Compounds 5–9
Yang et al.
5a
6a
7b
8c
9b
H-1
H-2a
H-2b
H-3
H-5
H-6
H-7
3.02 (m)
2.94 (m)
2.79 (m)
2.06 (m)
1.53 (m)
4.31 (br t, 7.8)
2.79 (m)
4.00 (br t, 10.0)
2.19 (ddd, 10.0, 10.0,
10.0)
2.69 (dd, 8.0, 8.5)
2.21 (td, 13.5, 8.0)
1.60 (td, 13.5, 8.0)
4.44 (br t, 8.0)
2.60 (dd, 10.0, 8.5)
3.95 (br t, 10.0)
1.68 (m)
2.80 (m)
2.27 (td, 13.5, 8.0)
1.94 (td, 13.5, 9.0)
4.77 (br t, 8.0)
3.13 (t, 10.0)
4.41 (br t, 10.0)
3.63 (m)
2.35 (td, 13.1, 6.0)
2.03 (td, 13.1, 8.5)
4.81 (br t, 6.0)
2.94 (m)
4.27 (br t, 10.0)
2.56 (ddd, 10.0, 10.0,
10.0)
2.08 (td, 13.0, 8.0)
1.53 (td, 13.0, 8.5)
4.31 (br t, 8.5)
2.80 (m)
4.00 (br t, 10.0)
2.19 (ddd, 10.0, 10.0,
10.0)
H-8
5.34 (m)
3.91 (m)
3.58 (m)
3.41 (m)
3.57 (m)
H-9a
H-9b
H-11
H-13
3.02 (m)
2.41 (dd, 12.6, 8.0)
3.06 (m)
3.67 (dd, 13.5, 6.0)/3.56
(d, 13.5)
2.94 (m)
2.47 (dd, 12.5, 7.3)
3.01 (m)
2.57 (m)
2.06 (m)
2.87 (m)
3.18 (m)/2.87 (m)
2.57 (dd, 12.0, 5.0)
1.71 (m)
2.89 (dt, 10.0, 3.0)
3.87 (m)/2.99 (dd, 12.5,
10.0)
2.55 (m)
2.04 (m)
2.89 (dt, 10.0, 3.5)
3.17 (dd, 12.0, 3.5)/2.55
(m)
3.98 (m)/3.12 (br t, 11.6)
H-14
H-15
H-3′
5.21 (s)/5.06 (s)
5.68 (s)/5.57 (s)
4.32 (d, 10.5) /4.11 (d,
10.5)
5.20 (s)/5.11 (s)
5.60 (s)/5.71 (s)
4.93 (s)/4.88 (s)
5.14 (s)/5.10 (s)
4.97 (s)/4.89 (s)
5.17 (s)/5.21 (s)
4.93 (s)/4.88 (s)
5.14 (d, 1.5)/5.10 (d, 1.5)
H-4′
1.70 (s)
H-2′′
H-3′′
H-4′′
H-5′′
3.79 (t, 7.0)
3.98 (m)
3.33 (2H, d, 6.1)
2.82 (m)
1.88 (m)
0.87 (3H, d, 6.6)
0.90 (3H, d, 6.6)
3.87 (t, 5.5)
3.41 (m)/3.87 (m)
2.96 (d, 5.0)
1.64 (m)
1.47 (m)/1.14 (m)
0.86 (d, 7.0)
2.21 (2H, m)
1.84 (m)/1.67 (m)
3.46 (dt, 10.0, 5.0) 2.76
(q, 10.0)
7.29 (2H, t, 7.3)
H-6′′
H-7′′
H-8′′
H-9′′
H-10′′
H-11′′
7.49 (2H, d, 7.3)
7.22 (t, 8.0)
7.49 (2H, d, 7.3)
7.29 (2H, t, 7.3)
7.39 (d, 8.0)
0.83 (d, 7.0)
7.07 (dt, 7.0, 1.0)
7.14 (dt, 7.0, 1.0)
7.69 (d, 8.0)
7.24 (s)
a Measured in pyridine-d5 at 500 MHz. b Measured in DMSO-d6 at 500 MHz. c Measured in CD3OD at 500 MHz.
Table 3. 13C NMR Data of Compounds 1-9
RP-18 column chromatography (20% CH3CN) to afford 4 (22 mg,
0.00063% w/w). Subfraction B223 (3.0 g) was subjected to Sephadex
LH-20 (70% MeOH) and a Si gel column (EtOAc-MeOH-H2O, 9:3:
1) and purified using preparative HPLC (EtOAc-MeOH-H2O, 9:3:1)
to afford 5 (60 mg, 0.0017% w/w). Subfraction B224 (4.0 g) was
subjected to Sephadex LH-20 column chromatography (70% MeOH)
and Lichroprep RP-18 column chromatography (50% MeOH) and
purified using preparative HPLC (EtOAc-MeOH-H2O, 9:3:0.5) to
afford 6 (8 mg, 0.00023% w/w). Subfraction B225 (1.0 g) was subjected
to Sephadex LH-20 column chromatography (70% MeOH), Lichro-
prepRP-18 column chromatography (50% MeOH), and Lichroprep Si
60 column chromatography (EtOAc-MeOH-H2O, 9:3:1) and purified
using preparative HPLC (20% CH3CN) to afford 7 (20 mg, 0.00057%
w/w). Subfraction B23 (6.0 g) was subjected to RP-18 column
chromatography (50% MeOH) to give three subfractions (B231-B232).
Subfraction B231 (3.0 g) was subjected to Sephadex LH-20 (70%
MeOH) and Lichroprep Si 60 column chromatography (EtOAc-
MeOH-H2O, 9:3:0.5) and purified using preparative HPLC (25%
CH3CN) to afford 8 (13 mg, 0.00037% w/w) and 9 (25 mg, 0.00071%
w/w).
1a
2b
3c
4d
5e
6e
7d
8b
9d
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
45.3 45.2 43.0 42.6 42.9 44.5 43.8 43.8 42.5
39.0 39.7 37.8 38.1 39.7 39.9 38.1 39.3 38.1
73.7 73.9 72.8 71.7 73.1 73.5 71.6 73.8 71.6
152.2 154.4 153.0 154.1 155.5 155.0 154.1 154.4 154.1
51.3 51.5 49.1 48.5 50.9 50.6 48.3 50.3 48.4
78.4 80.7 80.9 78.7 80.8 80.3 78.7 81.2 78.5
47.5 53.5 53.6 54.9 46.0 64.3 54.2 55.0 55.0
74.2 78.4 73.5 72.3 77.9 74.4 72.4 74.2 72.4
37.3 40.3 43.7 43.6 42.9 45.2 46.2 46.3 43.9
C-10 141.6 144.4 143.6 144.7 144.5 145.7 144.5 145.0 144.5
C-11 137.3 42.3 44.5 46.2 46.7 48.9 48.5 45.1 46.1
C-12 168.9 180.7 177.8 176.1 178.5 177.2 175.9 176.3 175.9
C-13 122.5 15.9 47.3 48.9 55.3 49.8 48.5 48.1 48.2
C-14 118.2 117.2 116.2 114.3 116.7 115.5 114.3 116.0 114.4
C-15 113.6 111.4 111.0 108.8 109.0 110.3 108.7 110.0 108.7
C-1′
C-2′
C-3′
C-4′
172.0 175.6
46.5 76.9
69.2 69.3
29.3 22.3
29.2
176.2
77.2
69.8
23.2
8r-O-(3′-Hydroxy-3′-methylbutyryl)desacylcynaropicrin (1): col-
C-5′
orless oil; [R]D +23.0 (c 0.3, MeOH); UV (MeOH) λmax (log ꢀ) 201
(3.09) nm; IR (neat) νmax 3401 (OH), 1766 (lactone), 1732 (ester) cm-1
;
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
C-6′′
C-7′′
C-8′′
C-9′′
C-10′′
C-11′′
174.5 174.4 177.2 176.4 173.6 173.6 173.2
59.3 47.9 68.8 64.3 67.6 63.7 66.4
33.9 24.3 30.5 40.2 22.0 26.4 36.9
172.8 31.9 25.1 138.8 19.2 108.3 24.9
55.5 130.5 18.3 128.8 11.5
1H and 13C NMR, see Tables 1 and 3; HREIMS m/z 362.1732 [M]+
(calcd for C20H26O6, 362.4228).
8r-O-(2′,3′-Dihydroxyisobutyryl)11ꢀ,13-dihydrodesacylcynaropi-
crin (2): colorless oil; [R]D +24.1 (c 0.1, MeOH); UV (MeOH) λmax
(log ꢀ) 201 (3.37) nm; IR (neat) νmax 3399 (OH), 1769 (lactone), 1735
129.1
127.3
129.1
130.5
122.9 15.5
120.4
119.5
112.9
138.3
(ester), 1644 (CdC) cm-1 1H and 13C NMR, see Tables 1 and 3;
;
HREIMS m/z 366.1679 [M]+ (calcd for C19H26H7, 366.4112).
Pulchellamine A (3): colorless gum; ninhydrin positive; [R]D +41.4
(c 0.3, MeOH); UV (MeOH) λmax (log ꢀ) 200 (3.61) nm; IR (neat) νmax
125.8
1
3338 (OH, COOH), 1762 (lactone), 1640 (CdC) cm-1; H and 13C
a Measured in CDCl3 at 125 MHz. b Measured in CD3OD at 125
MHz. c Measured in D2O at 125 MHz. d Measured in DMSO-d6 at 125
MHz. e Measured in pyridine-d5 at 125 MHz.
NMR, see Tables 1 and 3; HRESIMS m/z 395.1827 [M + H]+ (calcd
for C19H27N2O7H, 395. 1812).
Pulchellamine B (4): white crystals (obtained by recrystallization
from MeOH-H2O); mp 198 °C; ninhydrin positive; [R]D +52.0 (c 0.15,
MeOH); UV (MeOH) λmax (log ꢀ) 200 (3.80) nm; IR (neat) νmax 3297
Pulchellamine C (5): colorless gum; ninhydrin positive; [R]D +22.0
(c 0.5, MeOH); UV (MeOH) λmax (log ꢀ) 202 (3.94) nm; IR (neat) νmax
1
1
(OH, COOH), 1764 (lactone) cm-1; H and 13C NMR, see Tables 1
3305 (OH, COOH), 1755 (lactone), 1632 (CdC) cm-1; H and 13C
and 3; HRESIMS m/z 366.1927 [M + H]+ (calcd for C19H27NO6H,
NMR, see Tables 2 and 3; HRESIMS m/z 480.2249 [M + H]+ (calcd
366.1911).
for C24H33NO9H, 480.2228).

Guaiane Sesquiterpene Lactones from Saussurea pulchella
Journal of Natural Products, 2008, Vol. 71, No. 4 683
Acknowledgment. This research was supported by the Korea
Science and Engineering Foundation (KRF-2004-202-E00227). The
authors would like to thank Dr. E. K. Kwon, S. I. Lee, and Dr. J. J.
Seo at the Korea Basic Science Institute for the measurements of NMR
and MS spectra.
Table 4. Cytotoxicity of Compounds 1–15
ED50 valuesa
compound
A 549
SK-OV-3
SK-MEL-2
HCT 15
1
2
3
4
5
6
7
8
>30.0
>30.0
>30.0
>30.0
27.57
>30.0
>30.0
>30.0
>30.0
25.71
8.22
12.42
>30.0
>30.0
>30.0
11.83
>30.0
>30.0
24.19
>30.0
11.27
2.49
6.78
>30.0
>30.0
>30.0
5.73
>30.0
>30.0
18.56
>30.0
9.74
14.23
>30.0
>30.0
>30.0
18.33
>30.0
>30.0
>30.0
>30.0
11.33
3.82
12.13
>30.0
28.76
>30.0
0.164
Supporting Information Available: 1H, 13C, and 2D NMR (1H-1H
COSY, HMBC, HMQC, HSQC, NOESY) data for compounds 1 and
3. 1H NMR data for compounds 2 and 4-9. This material is available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
9
10
11
(1) Lee, S. O.; Choi, S. Z.; Choi, S. U.; Lee, K. Ch.; Chin, Y. W.; Kim,
J. W.; Kim, Y. Ch.; Lee, K. R. J. Nat. Prod. 2005, 68, 1471–1474.
(2) Choi, S. Z.; Kwon, H. Ch.; Choi, S. U.; Lee, K. R. J. Nat. Prod.
2002, 65, 1102–1106.
(3) Yin, H. Q.; Fu, H. W.; Hua, H. M.; Qi, X. L.; Li, W.; Sha, Y.; Pei,
Y. H. Chem. Pharm. Bull. 2005, 53, 841–842.
(4) Sun, Ch. M.; Syu, W. J.; Don, M. J.; Lu, J. J.; Lee, G. H. J. Nat.
Prod. 2003, 66, 1175–1180.
(5) Wang, H. B.; Zhang, H. P.; Zhou, Y.; Zuo, J. P.; Qin, G. W. J. Nat.
Prod. 2005, 68, 762–765.
(6) Matsuda, H.; Kageura, T.; Inoue, Y.; Morikawa, T.; Yoshikawa, M.
Tetrahedron 2000, 56, 7763–7777.
(7) Matsuda, H.; Toguchida, I.; Ninomiya, K.; Kageura, T.; Morikawa,
T.; Yoshikawa, M. Bioorg. Med. Chem. 2003, 11, 709–715.
(8) Li, S.; An, T.-Y.; Li, J.; Shen, Q.; Lou, F.-C.; Hu, L.-H. J. Asian Nat.
Prod. Res. 2006, 8, 281–286.
(9) Duan, H.; Takaishi, Y.; Momota, H.; Ohmoto, Y.; Taki, T. Phy-
tochemistry 2002, 59, 85–90.
(10) Takasaki, M.; Konoshima, T.; Komatsu, K.; Tokuda, H.; Nishino, H.
Cancer Lett. 2000, 158, 53–59.
(11) Fan, Ch. Q.; Yue, J. M. Bioorg. Med. Chem. 2003, 11, 703–708.
(12) Xie, H.; Wang, T.; Matsuda, H.; Morikawa, T.; Yoshikawa, M.; Tani,
T. Chem. Pharm. Bull. 2005, 53, 1416–1422.
(13) Kushnir, L. E.; Kuzovkov, A. D. Khim. Prir. Soedin. 1966, 2, 245–
248.
(14) Kushnir, L. E.; Kuzovkov, A. D. Khim.-Farm. Zh. 1968, 2, 21–29.
(15) Agafonova, N. V.; Kushnir, L. E.; Kuzovkov, A. D.; Shreter, A. I.;
Pimenov, M. G. Aptechnoe Delo 1966, 15, 36–37.
(16) Basargin, D. D.; Tsiklauri, G. Ch. Rastit. Resur. 1990, 26, 68–71.
(17) An, D. K. Illustrated Book of Korean Medicinal Herbs; Kyohaksa:
Seoul, 1998; p 164.
(18) Yang, M. C.; Lee, K. H.; Kim, K. H.; Choi, S. U.; Lee, K. R. Arch.
Pharm. Res. 2007, 30, 1067–1074.
(19) Ha, T. J.; Jang, D. S.; Lee, J. R.; Lee, K. D.; Lee, J.; Hwang, S. W.;
Jung, H. J.; Nam, S. H.; Park, K. H.; Yang, M. S. Arch. Pharm. Res.
2003, 26, 925–928.
(20) Rustaiyan, A.; Niknejad, A.; Zdero, C.; Bohlmann, F. Phytochemistry
1981, 20, 2427–2429.
(21) Bohlmann, F.; Singh, P.; King, R. M.; Robinson, H. Phytochemistry
1982, 21, 1171–1172.
(22) Choi, S. Z.; Choi, S. U.; Lee, K. R. Arch. Pharm. Res 2005, 28, 1142–
1146.
(23) Tan, R. X.; Jakupovic, J.; Bohlmann, F.; Jia, Z. J.; Schuster, A.
Phytochemistry 1990, 29, 1209–1212.
(24) Singhal, A. K.; Chowdhury, P. K.; Sharma, R. P.; Baruah, J. N.; Herz,
W. Phytochemistry 1982, 21, 462–463.
(25) Navarro, J. J.; Caballero, M. C.; Moran, J. R.; Medarde, M.; Grande,
M.; Anaya, J. J. Nat. Prod. 1990, 53, 573–578.
(26) Shimizu, Sh.; Ishihara, N.; Umehara, K.; Miyase, T.; Ueno, A. Chem.
Pharm. Bull. 1988, 36, 2466–2474.
(27) Seaman, F. C.; Fischer, N. H. Phytochemistry 1980, 19, 583–586.
(28) Yoshikawa, M.; Hatakeyama, Sh.; Inoue, Ya.; Yamahara, J. Chem.
Pharm. Bull. 1993, 41, 214–216.
(29) Malmstrom, J.; Ryager, A.; Anthoni, U.; Nielsen, P. H. Phytochemistry
2002, 60, 869–872.
(30) Malmstrom, J.; Ryager, A.; Anthoni, U.; Nielsen, P. H. Phytochemistry
1999, 62, 787–789.
1.53
4.07
12
13
14
15
24.51
>30.0
>30.0
>30.0
0.007
7.42
>30.0
18.04
>30.0
0.056
>30.0
10.52
>30.0
0.117
doxorubicin
a ED50 value of compounds against each cancer cell line, which was
defined as a concentration (µM) that caused 50% inhibition of cell
growth in Vitro.
Pulchellamine D (6): colorless gum; ninhydrin positive; [R]D +74.0
(c 0.3, MeOH); UV (MeOH) λmax (log ꢀ) 202 (3.97) nm; IR (neat) νmax
3333 (OH, COOH), 1763 (lactone), 1638 (CdC) cm-1; H and 13C
1
NMR, see Tables 2 and 3; HRESIMS m/z 428.2080 [M + H]+ (calcd
for C24H29NO6H, 428.2067).
Pulchellamine E (7): white crystals (obtained by recrystallization
from MeOH-H2O); mp 220 °C; ninhydrin positive; [R]D +29.0 (c 0.2,
MeOH); UV (MeOH) λmax (log ꢀ) 202 (3.89) nm; IR (neat) νmax 3333
(OH, COOH), 1765 (lactone), 1636 (CdC) cm-1; H and 13C NMR,
1
see Tables 2 and 3; HRESIMS m/z 380.2067 [M + H]+(calcd for
C20H29NO6H, 380.2068).
Pulchellamine F (8): colorless gum; ninhydrin positive; [R]D +40.4
(c 0.4, MeOH); UV (MeOH) λmax (log ꢀ) 202 (3.45), 221 (3.49), 282
(2.77) nm; IR (neat) νmax 3301 (OH, COOH), 1762 (lactone), 1634
1
(CdC) cm-1; H and 13C NMR, see Tables 2 and 3; HRESIMS m/z
467.2190 [M + H]+ (calcd for C26H30N2O6H, 467.2176).
Pulchellamine G (9): white crystals (obtained by recrystallization
from MeOH-H2O); mp 199 °C; ninhydrin positive; [R]D +15.4 (c 0.1,
MeOH); UV (MeOH) λmax (log ꢀ) 203 (4.00) nm; IR (neat) νmax 3372
(OH, COOH), 1766 (lactone), 1633 (CdC) cm-1; H and 13C NMR,
1
see Tables 2 and 3; HRESIMS m/z 394.2214 [M + H]+(calcd. for
C21H31NO6H, 394.2224).
6
Synthesis of Pulchellamines A-G (3-9). A solution of 10 (2.0
mg, 0076 mmol) in EtOH (1.0 mL) was treated with amino acid {L-
asparagine (2.0 mg, 0.015 mmol: in 3), 4-aminobutanoic acid (0.8 mg,
0.0076 mmol: in 4), L-phenylalanine (3.8 mg, 0.023 mmol: in 6),
L-valine (1.8 mg, 0.015 mmol: in 7), L-tryptophan (6 mg, 0.03 mmol:
in 8), and L-isoleucine (2.6 mg, 0.02 mmol: in 9)} in the presence of
Et3N (0.05 mL), and the mixture was heated under reflux for 1 h. In
the synthesis of 5, a solution of 14 (2.0 mg, 0.0076 mmol) in EtOH
(1.0 mL) was stirred at room temperature for 72 h with L-proline (1.7
mg, 0.015 mmol). After cooling, the reaction mixture was evaporated
under reduced pressure and the residue purified by using preparative
HPLC (EtOAc-MeOH-H2O, 9:3:0.5, 9:3:1) to give compounds 3-9
(3: 0.4 mg, 13%, 4: 0.4 mg, 14%, 5: 1.5 mg, 57%, 6: 2.0 mg, 61%, 7:
0.7 mg, 24%, 8: 2.2 mg, 62%, 9: 2.0 mg, 67%).
Test for Cytotoxicity in Vitro. A sulforhodamin B bioassay (SRB)
was used to determine the cytotoxicity of each compound against four
cultured human cancer cell lines.31 The assays were performed at the
Korea Research Institute of Chemical Technology. The cell lines used
were A549 (non small cell lungcarcinoma), SK-OV-3 (ovary malignant
ascites), SK-MEL-2 (skin melanoma), and HCT (colon adenocarci-
noma). Doxorubicin was used as a positive control. The cytotoxicities
of doxorubicin against A549, SK-OV-3, SK-MEL-2, and HCT cell lines
were ED50 0.007, 0.056, 0.117, and 0.164 µM, respectively.
(31) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
J. Natl. Cancer Inst. 1990, 82, 1107–1112.
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