Four new cytotoxic oligosaccharidic derivatives of 12-oleanene from Lysimachia heterogenea Klatt

Article abstract of DOI:10.1016/j.bmcl.2009.10.056

Cytotoxicity-guided phytochemical analysis on the extract of Lysimachia heterogenea Klatt led to the isolation of 3β,16β-12-oleanene-3,16,23,28-tetrol (1) and its four new oligosaccharidic derivatives heterogenosides A, B, C, and D (2-5). Their structural

Full text of DOI:10.1016/j.bmcl.2009.10.056

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6515–6518
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Four new cytotoxic oligosaccharidic derivatives of 12-oleanene
from Lysimachia heterogenea Klatt
b
c
a
d
b
Xin-an Huang a, , Yong-ju Liang , Xiao-ling Cai , Xiao-quan Feng , Chuan-hai Zhang , Li-wu Fu ,
*
Wen-di Deng a
a Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China
b State Key Laboratory for Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou 510060, China
c School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
d Department of Chemistry and Biology, West Anhui University, Lu-an 237012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytotoxicity-guided phytochemical analysis on the extract of Lysimachia heterogenea Klatt led to the iso-
lation of 3b,16b-12-oleanene-3,16,23,28-tetrol (1) and its four new oligosaccharidic derivatives hetero-
genosides A, B, C, and D (2–5). Their structural elucidation was mainly based on NMR and mass
spectral data. The time course experimental results indicated that unlike the likely lysis activity of het-
erogenosides B–D, heterogenoside A showed a significantly time-dependent cytotoxicity.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 29 June 2009
Revised 13 September 2009
Accepted 13 October 2009
Keywords:
Lysimachia heterogenea Klatt
Cytotoxicity
Oligosaccharidic derivatives of 12-oleanene-
3,16,23,28-tetrol
The genus Lysimachia, traditionally classified in the family Prim-
ulaceae, was newly assigned into the Myrsinaceae on basis of
molecular phylogenetic analysis.1 The genus comprises more than
193 species, over 90% of which are endemic to China.2 Over thirty
new triterpenoid sapponins had been isolated from this genus in
the last five years.3 Lysimachia heterogenea Klatt, an endemic spe-
cies, is a perennial herb, which was used as a folk medicine in sub-
duing swelling and detoxicating in China.4 As a part of the
continuous investigation on the chemical components in this
genus,5 the human lung cancer (A549) cell line was used in cyto-
toxicity-guided phytochemical analysis, by which the effective
fraction LH-1 of L. heterogene was found. The further investigation
on the antitumor components of the fraction LH-1 led to the isola-
tion of 3b,16b-12-oleanene-3,16,23,28-tetrol (1) and its four new
oligosaccharidic derivatives heterogenosides A–D (2–5). In this
Letter, the structure and the antitumor activity of these four new
compounds was reported.
D101 resin column using 20% and 90% EtOH solvents as eluant,
respectively. The latter eluate (90.6 g), with IC50 value of 4.0 g/
mL, was further chromatographed on a silica gel column to obtain
fractions LH-1 and LH-2 using 20% and 70% MeOH/CHCl3 solvents
as eluant, respectively. Fractions LH-1 and LH-2 presented the
l
cytotoxicity with the IC50 values of 2.5 and 45.0 lg/mL, respec-
tively. Therefore, Fraction LH-1 was served as the effective antitu-
mor fraction of L. heterogenea, and it was further purified by silica
gel chromatography to yield 12-oleanene-3,16,23,28-tetrol
(MeOH–CHCl3, 5:95, v/v, 8 mg, 1), heterogenoside A (MeOH–CHCl3,
10:90, v/v, 15 mg, 2), heterogenoside B (MeOH–CHCl3, 15:85, v/v,
20 mg, 3), heterogenoside C (MeOH–CHCl3, 15:85, v/v, 40 mg, 4),
and heterogenoside D (MeOH–CHCl3, 22:78, v/v, 13 g, 5).
Heterogenoside A (2) was obtained as white powder (MeOH).
The HRESIMS implied that 2 had the molecular C41H68O13. The IR
spectrum showed the presence of hydroxyl groups due to
absorption bands at 3390, 1077, and 1043 cmꢀ1. The 1H NMR
spectrum displayed six quaternary methyl signals at d 0.94,
0.99, 1.00, 1.02, 1.10, and 1.78, as well as an olefinic proton sig-
nal at d 5.40 (Table 1). The HMBC correlations from H-24 to C-3,
C-4, C-5, and C-23; H-25 to C-10; H-26 to C-7, C-8, C-9, and
C-14; H-27 to C-14; H-29 to C-19, C-20, C-21, and C-30; H-30
to C-19, C-20, C-21, and C-29 established the aglycon to be
The dried L. heterogenea (1.70 kg) were powered, then extracted
with 95% EtOH for 24 h at room temperature for three times to give
the crude extract 180.0 g. The crude extract was sequentially par-
titioned with petroleum ether, EtOAc, and n-BuOH, respectively.
Among them the n-BuOH fraction (100.2 g) exhibited cytotoxicity
with IC50 value of 4.3 lg/mL, then it was chromatographed on a
3b,16b-12-oleanene-3,16,23,28-tetrol. The sugar residues of
2
were determined to be arabinose and glucose after hydrolysis
by co-TLC with authentic sugars. The coupling constant analysis
* Corresponding author.
E-mail address: xahuang@163.net (X. Huang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.056

6516
X. Huang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6515–6518
Table 1
The main 1H NMR data for compounds 2–5 (500 MHz in pyridine-d5)
No.
1
2
3
4
5
1.09a
1.12a
1.04a
1.11a
1.64 (m)
2.02 (m)
2.23a
1.64 (m)
2.02 (m)
2.22a
1.59a
1.58a
2
1.95a
1.95a
2.09 (m)
4.11 (m)
1.60a
2.09 (m)
4.10 (m)
1.60a
3
5
6
4.24a
4.25a
1.68a
1.69a
1.38a
1.38a
1.35a
1.35a
1.69a
1.70a
1.69a
1.69a
7
1.32a
1.35a
1.30a
1.31a
1.78a
1.78a
1.72a
1.74a
9
1.89 (br s)
1.95 (m)
5.39 (br s)
1.58 (m)
2.19a
1.91 (br s)
1.95 (m)
5.39 (br s)
1.59 (m)
2.20a
1.86 (m)
1.90 (m)
5.39 (br s)
1.58a
1.88 (br s)
11
12
15
1.92a
5.38 (br s)
1.58a
2.19 (m)
2.18 (m)
16
18
19
4.60a
4.61a
4.59a
4.59a
2.49 (dd, 15.0, 1.5 Hz)
2.49 (dd, 15.0, 1.5 Hz)
2.49 (dd, 15.0, 1.5 Hz)
1.31a
2.48 (dd, 15.0, 1.5 Hz)
1.30a
1.32a
1.32a
2.72 (t, 15.0 Hz)
1.43 (m)
2.38 (td, 12.5, 4.5 Hz)
1.38a
2.72 (t, 15 .0 Hz)
1.43 (m)
2.38 (td, 12.5, 4.5 Hz)
1.38a
2.71 (t, 15 .0 Hz)
1.42 (m)
2.38 (td, 12.5, 4.5 Hz)
1.35a
2.70 (t, 15 .0 Hz)
1.42 (m)
2.38 (td, 12.5, 4.5 Hz)
1.35a
2.25 (td, 12.5, 4.5 Hz)
3.70 (d, 11.5 Hz)
4.28a
1.08 (s)
0.98 (s)
0.96 (s)
1.75 (s)
21
22
23
a
a
2.28
2.28
2.25 (td, 12.5, 4.5 Hz)
3.68a
3.71a
3.71a
4.27a
4.35a
4.18 (d, 11.0 Hz)
1.02 (s)
0.98 (s)
0.96 (s)
1.74 (s)
3.61 (d, 11.5 Hz)
3.73 (d, 11.5 Hz)
1.02 (s)
24
25
26
27
28
0.94 (s)
1.00 (s)
0.99 (s)
1.77 (s)
3.62 (d, 11 Hz)
3.74 (d, 11 Hz)
1.02 (s)
1.00 (s)
1.00 (s)
1.00 (s)
1.77 (s)
3.61 (d, 11.5 Hz)
3.73 (d, 11.5 Hz)
1.03 (s)
3.61 (d, 11.5 Hz)
3.73 (d, 11.5 Hz)
1.03 (s)
29
30
1.11 (s)
1.12 (s)
1.12 (s)
1.11 (s)
Arabinose
1
2
3
4
5
4.92 (d, 7.5 Hz)
4.86 (d, 7.5 Hz)
4.07 (br s)
4.32a
5.12 (d, 8.0 Hz)
4.54 (m)
4.34 (m)
4.42a
5.00 (d, 5.5 Hz)
4.57a
4.30a
4.40a
4.18 (m)
4.22a
4.21a
4.21a
3.67 (dd, 10.0, 0.5 Hz)
3.63 (dd, 10.0, 0.5 Hz)
3.72a
3.72a
4.40a
4.61a
4.57a
4.57a
Glucose (at C-2 of arabinose)
1
2
3
4
5
6
5.24 (d, 8.0 Hz)
5.01 (d, 7.5 Hz)
3.97a
5.14 (d, 7.5 Hz)
4.05a
5.49 (d, 7.0 Hz)
4.05a
4.16 (t, 7.5 Hz)
4.24a
4.25a
4.22 (m)
4.22 (m)
4.25a
4.27a
4.22a
4.22a
3.91 (br s)
3.82 (br s)
3.87a
3.78a
4.29a
4.34a
4.35a
4.35a
4.51 (dd,11.0, 1.0 Hz)
4.47 (d,11.0 Hz)
4.47(d,11.0 Hz)
4.41a
Glucose (at C-4 of arabinose)
1
2
3
4
5
6
5.14 (d, 7.5 Hz)
4.05a
5.00 (d, 7.5 Hz)
3.94 (m)
4.19a
4.22 (m)
4.21a
4.22a
3.87a
3.87a
4.35a
4.35a
4.47 (d,11.0 Hz)
4.41a
Xylose
1
2
3
4
5
4.90 (d, 6.5 Hz)
3.98a
4.96 (d, 6.0 Hz)
4.11 (m)
4.00a
3.97a
3.98a
4.17a
3.48 (m)
3.72a
4.28a
4.58a
a
The signals were overlapped.
of the anomeric protons at
(d, J = 7.5 Hz) supported the
b-
to C-3 and H00-1 to C0-2 were used to determine the oligosaccha-
ride as 3-O-b- -glucopyranosyl-(1?2)- -arabinopyranosyl moi-
ety. On the basis of above findings, the structure of 2 was
d
4.92 (d, J = 7.5 Hz) and 5.24
-configuration of arabinose and
concluded to be 3-O-[b-
anosyl]-3b,16b-12-oleanene-3,16,23,28-tetrol (Fig. 1).
Heterogenoside B (3) was isolated as white powder (MeOH).
D-glucopyranosyl-(1?2)-a-L-arabinopyr-
a
-
L
D
-configuration of glucose. The HMBC correlations from H0-1
The HRESIMS revealed its molecular formula to be C46H76O17
.
D
a-
L
The 13C NMR data of 3 were similar to that of 2, except 3 contained
an additional pentose unit (Table 2). The hydrolysis analysis and

X. Huang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6515–6518
6517
(1?4)-[b-
Thus, 4 was elucidated as 3-O-{b-
glucopyranosyl-(1?2)]- -arabinopyranosyl}-3b,16b-12-olean-
D
-glucopyranosyl-(1?2)]-
a-
L-arabinopyranosyl moiety.
D-glucopyranosyl-(1?4)-[b-D-
a
-L
ene-3,16,23,28-tetrol (Fig. 1).
Heterogenoside D (5) presented as white powder (MeOH). The
HRESIMS gave its molecular formula to be C52H86O22. The 13C
NMR data of the aglycon of 5 was identical to that of 2, 3, and 4 (Ta-
ble 2). Compared with the HRESIMS data of 4, 5 comprised an addi-
tional pentose unit which was ascertained to be b-D-xylose by
comparing the hydrolysis products with the authentic sugar and
analyzing the coupling constant of the anomeric proton at d 4.96
(d, J = 6.0 Hz) (Table 1). The glycosidic linkages between monosac-
charide residues were defined as 3-O-b-
D
-xylopyranosyl-(1?2)-b-
D
-glucopyranosyl-(1?4)-[b-
D
-glucopyranosyl-(1?2)]- -arabino-
a-L
pyranosyl due to the long-range correlations from H0-1 to C-3 and
C0-5, H00-1 to C0-2, H000-1 to C0-4, H000-2 to C000-1 and C000-3, H0000-1 to C000-
2, H0000-5 to C0000-1, C0000-3 and C0000-4. Therefore, 5 was structurally
Figure 1. The structures of compounds 1–5.
explained to be 3-O-{b-
syl-(1?4)-[b- -glucopyranosyl-(1?2)]-
16b-12-oleanene-3,16,23,28-tetrol (Fig. 1).
D
-xylopyranosyl-(1?2)-b-
D-glucopyrano-
D
a-L
-arabinopyranosyl}-3b,
the coupling constant of the anomeric proton at
d 4.90
In addition to the above four new compounds, the known com-
pound of 3b,16b-12-oleanene-3,16,23,28-tetrol was identified by
comparing its spectral data (Table 2) with the literature.6
The half-inhibitory concentration (IC50) values of heterogeno-
sides A–D were measured after 2, 4, 9, 18, 36, and 72 h of
incubation with the A549 cells, respectively. The activity of hetero-
genosides B–D slightly increased during continuous incubation,
(d, J = 6.5 Hz) confirmed the pentose as b-
HMBC correlations from H0-1 to C-3, H00-1 to C0-2, H000-1 to C00-2,
H00-2 to C00-1 and C000-1 established the 3-O-b-
-xylopyranosyl-
(1?2)-b- -glucopyranosyl-(1?2)- -arabinopyranosyl moiety.
Hence, the structure of 3 was identified as 3-O-[b- -xylopyrano-
syl-(1?2)-b- -glucopyranosyl-(1?2)- -arabinopyranosyl]-3b,
16b-12-oleanene-3,16,23,28-tetrol (Fig. 1).
D-xylose (Table 1). The
D
D
a-L
D
D
a-L
their IC50 values were calculated to be 26.4, 23.6, and 20.2
lM at
Heterogenoside C (4) appeared as white powder (MeOH). The
HRESIMS determined its molecular formula to be C47H78O18. Com-
pared with the 13C NMR and HRESIMS spectrum of 2, 4 differed in
the additional hexose unit (Table 2). The analysis of the hydrolysis
products and the coupling constant of the anomeric proton at d
the end of 2 h of incubation, and 19.1, 16.3, and 14.5 M at the
l
end of 72 h of incubation, respectively. Unlike heterogenosides
B–D, heterogenoside A showed weaker activity with IC50 value of
more than 100.0 lM even after 9 h of incubation, and 24.5 lM at
the end of 72 h of incubation. The time course experiment indi-
cated that the activity of heterogenoside A was significantly
time-dependent, while the activity of heterogenosides B–D was
likely due to lysis.
5.14 (d, J = 7.5 Hz) inferred the hexose sugar was b-D-glucose
(Table 1). The HMBC correlations from H-3 to C0-1, H0-1 to C-3,
C0-2 and C0-5, H0-2 to C0-1, C0-3 and C00-1, H00-1 to C0-2, H000-1 to
C0-4 deduced that C-3 was linked with b-
D-glucopyranosyl-
Table 2
13C NMR data for compounds 1–5 (125 MHz in pyridine-d5)
No.
1
2
3
4
5
No.
2
3
4
5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
39.0
27.7
73.6
42.8
48.8
18.6
33.0
40.1
47.2
37.1
23.9
121.2
148.2
42.0
34.8
74.2
40.9
42.5
48.3
31.2
37.1
30.4
68.3
13.1
16.2
17.1
27.3
70.1
33.4
24.8
38.9
26.1
82.2
43.5
47.7
18.2
32.9
40.1
47.2
36.9
23.9
122.3
145.2
42.0
34.8
74.2
41.0
42.6
48.4
31.2
37.1
30.5
64.6
13.6
16.4
17.1
27.3
70.2
33.4
24.9
38.9
26.0
82.2
43.5
47.8
18.2
32.9
40.1
47.2
36.9
23.9
122.3
145.2
42.0
34.8
74.2
40.9
42.5
48.2
31.2
37.1
30.4
64.7
13.6
16.4
17.1
27.3
70.1
33.4
24.8
38.9
25.9
82.3
43.5
48.0
18.2
32.9
40.1
47.2
36.8
23.9
122.3
145.2
42.0
34.8
74.2
40.9
42.5
48.3
31.2
37.1
30.4
64.9
13.4
16.4
17.1
27.3
70.1
33.4
24.8
38.9
25.8
82.5
43.6
48.1
18.2
32.9
40.1
47.2
36.9
23.9
122.3
145.2
42.0
34.8
74.2
40.9
42.5
48.3
31.2
37.1
30.4
65.0
13.4
16.3
17.1
27.3
70.1
33.4
24.9
Arabinose
1
2
3
4
5
106.3
79.7
73.7
74.6
66.3
106.5
81.2
73.9
74.5
66.5
103.5
81.2
72.7
77.2
63.7
104.1
80.3
73.4
78.4
64.3
Glucose (at C-2 of arabinose)
1
2
3
4
5
6
106.7
75.8
78.7
71.3
78.4
62.6
105.3
86.2
77.6
71.0
78.3
62.3
105.6
75.7
78.3
71.3
78.1
62.5
105.1
76.1
78.3
71.5
78.3
62.7
Glucose (at C-4 of arabinose)
1
2
3
4
5
6
105.7
76.1
78.6
71.4
78.1
62.6
103.7
85.3
77.6
71.1
77.8
62.3
Xylose
1
2
3
4
5
108.0
76.3
78.0
70.4
67.2
107.6
76.2
78.1
70.7
67.4

6518
X. Huang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6515–6518
Rydin, C.; Källersjö, M. Am. J. Bot. 2002, 89, 677; (d) Hao, G.; Yuan, Y. M.; Hu, C.
Acknowledgments
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We thank the National Natural Science Foundation of China
(30500052) for financial support. The work was also supported
by the National Key Discipline of Clinical Foundation of Chinese
Medicine.
Supplementary data
5. (a) Huang, X. A.; Yang, R. Z.; Cai, X. L.; Ye, S.; Hu, Y. J. J. Mol. Struct. 2007, 830, 100;
(b) Huang, X. A.; Ha, C. Y.; Yang, R. Z.; Jiang, H. Y.; Hu, Y. J.; Zhang, Y. H. Chem.
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Chin. Mat. Med. 2007, 32, 596.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.056.
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