全 文 ： 2012 年 1 月 第 10 卷 第 1 期 Chin J Nat Med Jan. 2012 Vol. 10 No. 1 13

Chinese Journal of Natural Medicines 2012, 10(1): 0013−0015
doi: 10.3724/SP.J.1009.2012.00013
Chinese
Journal of
Natural
Medicines






·Original papers·
A new carbamate with cytotoxic activity from the aerial
parts of Siegesbeckia pubecens
LIU Jia1, 3, CHEN Rong3, NIE Yu2, FENG Lin3, LI Hai-Dao3, LIANG Jing-Yu1*
1Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China;
2Department of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;
3Jiangsu Simcere Pharmaceutical R&D Co., Ltd., Nanjing 210042, China
Available online 20 Jan. 2012
[ABSTRACT] AIM: To study the chemical constituents of Siegesbeckia pubescens. METHODS: The chemical constituents were
isolated by extraction, crystallization and various chromatographic methods, and the chemical structures were elucidated on the basis of
spectral analysis. In addition, the cytotoxic activity of compound 1 was evaluated using human lung cancer cell A 549. RESULTS:
Four compounds were obtained, and their structures were identified as (E)-3-(3-oxobut-1-enyl)phenyl dimethylcarbamate (1),
ent-2-oxo-15, 16, 19-trihydroxypimar-8(14)-ene (2), 16-acetylkirenol (3), 3, 7-dimethylquercetin (4). CONCLUSION: Compound 1 is
a new carbamate, and the IC50 in MTT method of compound 1 was 58 μg·mL-1.
[KEY WORDS] Siegesbeckia pubescens; Chemical constituents; Cytotoxic activity
[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2012)01-0013-03

1 Introduction
Plants of the genus Siegesbeckia are annual herbs widely
distributed in tropical, subtropical, and temperate parts of the
world. Three species of this genus grow in China (Sieges-
beckia pubescens, S. orientalis, S. glabrescens), the aerial
parts of which have been used as traditional Chinese medi-
cines called “Xi-Xian”, to treat rheumatic arthritis, hyperten-
sion, malaria and snakebite[1]. The medicinal importance and
diverse activities of members of this genus promoted us to
undertake further phytochemical investigation of Sieges-
beckia pubescens. As a result , a new carbamate (1), along
with 3, 7-dimethylquercetin (2), and ent-2-oxo-15, 16,
19-trihydroxypimar-8(14)-ene (3), 16-acetylkirenol (4) were
afforded. This paper described the isolation and structural
elucidation of the new compound. Cytotoxic evaluation of
the new compound showed that compound 1 exhibited activ-
ity against human lung cancer cell A 549.

[Received on] 14-Jan.-2011
[Research funding] This project was supported by the Natural Sci-
ence Foundation of Jiangsu Province of China (No. BK2009470).
[*Corresponding author] LIANG Jing-Yu: Prof., E-mail: jyli-
ang08@126.com
These authors have no any conflict of interest to declare.
2 Experimental
2.1 General
Silica gel (48−75 μm) used for column chromatography
(CC) and silica GF254 (10−40 μm) for TLC were both sup-
plied by Qingdao Marine Chemical Plant, Qingdao, China;
Sephadex LH-20 was purchased from GE Healthcare
Bio-Sciences AB; and MCI gel was from Mitsubishi Chemi-
cal Corporation, Tokyo, Japan. 1H-, 13C-, and 2D-NMR
Spectra: Bruker-AV-500, 300 spectrometers; δ to Me4Si, J in
Hz. Mass spectra were obtained on a MS Agilent 1100
JC/MSD Trap (ESI-MS-MS) spectrometer and a Micro
Q-TOF MS (HRESI-MS), respectively.
2.2 Plant material
The aerial parts of Siegesbeckia pubescens were pur-
chased from the Bozhou Bohua Pharmaceutical Co., Ltd., in
Anhui Province, China (December 2009), and identified by
SONG Xue-Hua, the curator of China Pharmaceutical Uni-
versity (China). A voucher specimen has been deposited in
Department of Natural Medicinal Chemistry, China Pharma-
ceutical University, Nanjing, China.
2.3 Extraction and isolation
The air-dried and powdered Siegesbeckia pubescens
(10 kg) was extracted with 95% EtOH. The crude extract was
mixed with H2O (5 L) to form a suspension and then parti-
tioned successively with petroleum ether, EtOAc, and
LIU Jia, et al. /Chinese Journal of Natural Medicines 2012, 10(1): 13−15
14 Chin J Nat Med Jan. 2012 Vol. 10 No. 1 2012 年 1 月 第 10 卷 第 1 期

n-BuOH. The EtOAc-soluble part (130 g) was subjected to
MCI column eluted with EtOH- H2O (1 : 10, 4 : 6, 3 : 1, 10 :
1) to give four fractions (A-D). Fraction B was separated by
ODS column, using aceone-H2O (1 : 2, 1 : 1). Each subfrac-
tion of faction B was subjected to repeated silica gel,
Sephadex LH-20 column chromatography, and further puri-
fied by recrystallization. At last, compounds 1 (60 mg), 2 (10
mg), 3 (21 mg), and 4 (12 mg) were isolated.
3 Structural Identification
Compound 1 Viscous brown transparent oil. The mo-
lecular formula of 1 was determined as C13H15NO3 on the
basis of the ion peak m/z 256.094 8 [M + Na] + in HR-TOF-
MS (Calcd. for C13H15NO3Na; 256.095 0), which confirmed
by the 1H NMR, 13C NMR and HSQC. The 1H NMR (Table 1)
showed four meta-coupled aromatic protons at δ 7.16 (1H, dt,
J = 7.0, 2.2 Hz, H-6), 7.32 (1H, m, H-2), 7.36 (1H, m, H-4)
and 7.37 (1H, m, H-5), one methyl singlet signals at δ 2.37
(1H, s, H-4’’), and a trans double bond signal at δ 6.70 (1H, d,
J = 16.3 Hz, H-2’’), 7.47 (1H, d, J = 16.3 Hz, H-1’’). The 13C
NMR displayed six aromatic carbons at δ 121.1 (C-2), 123.8
(C-6), 125.2 (C-4), 129.7 (C-5), 135.8 (C-3), 152.0 (C-1),
two double bond carbons at δ 127.4 (C-2’’), 142.4 (C-1’’),
and one carbonyl carbon at δ 198.0 (C-3’’).The 1H NMR
signals at δ 3.02 (1H, s, H-2’) and 3.11 (1H, s, H-3’) and the
13C NMR signals at δ 36.4 (C-2’), 36.7 (C-3’) and 154.5
(C-1’), indicated a dimethylcarbamate group at C-1[2-3]. The
position of the double bond in 1 was evidenced by HMBC
(Fig. 1) correlations. The correlation between aromatic proton
δ 6.70 and δ 135.8 suggested that double bond positioned at
C-3. In addition, the double bond protons at δ 6.70 and 7.47
showed correlations with carbons at δ 198.0 and 27.6, indi-
cating that the acetyl was connected with the double bond.
Therefore, the structure of 1 was determined as
(E)-3-(3-oxobut-1-enyl) phenyl dimethylcarbamate. The
complete NMR data are listed in Table 1.

Table 1 NMR spectral data for compound 1 (500 MHz for 1H NMR and 125 MHz for 13C NMR in CDCl3)
HMBC
No. δH δC HSQC 2J 3J
1 – 152.0 -C- – –
2 7.32 m 121.1 -CH- C-1 C-4, 6, 1
3 - 135.8 -C- – –
4 7.36 m 125.2 -CH- C-3 C-2, 6, 1
5 7.37 m 129.7 -CH- C-6 C-1, 3
6 7.16 dt (7.0, 2.2) 123.8 -CH- C-1 C-2, 4
1 – 154.5 -C- – –
2 3.11 s 36.4 CH3 – C-1, 3
3 3.02 s 36.7 CH3 – C-1, 2
1 7.47 d (16.3) 142.4 -CH- C-3, 2 C-2, 4, 3
2 6.70 d (16.3) 127.7 -CH- C-1, 3 C-3,4
3 – 198.0 -C- – –
4 2.37 s 27.6 CH3 C-3 C-2

Compound 2 White powder, ESI-MS m/z 337 [M + H]
+, 359 [M + Na] +. 1H NMR (500 MHz, CDCl3) δ: 5.19 (1H,
br. s, H-14), 3.71 (1H, m, H-16a), 3.39 (1H, d, J = 11.0 Hz,
H-19a), 1.14 (3H, s, H-18), 0.90 (3H, s, H-17), 0.80 (3H, s,
H-20). 13C NMR (500 MHz, CDCl3) δ: 212.1 (C-2), 138.1
(C-8), 128.8 (C-14), 76.4 (C-15), 65.5 (C-19) 63.3 (C-16),
55.1 (C-5), 53.8 (C-1), 50.6 (C-9), 50.3 (C-3), 44.5 (C-10),
37.3 (C-12), 27.2 (C-18), 23.0 (C-17), 22.6 (C-6), 18.6
(C-11), 16.2 (C-20). Compound 2 was characterized as
ent-2-oxo-15, 16, 19-trihydroxypimar-8 (14)-ene by com-
parison of the physical and spectral data with the reported
data[4].
Compound 3 Pale yellow oil, ESI-MS m/z 381 [M + H]
+, 403 [M + Na] +, C22H36O5. 1H NMR (500 MHz, CDCl3) δ:
5.16 (1H, br s, H-14), 4.25 (1H, dd, J = 11.4, 2.4 Hz, H-16a),
4.05 (1H, dd, J = 11.4, 9.0 Hz, H-16b), 3.83 (1H, m, H-2),
3.71 (1H, d, J = 10.9 Hz, H-19a), 3.71 (1H, dd, J = 9.0, 2.4
Hz, H-15), 3.42 (1H, d, J = 10.9 Hz, H-19b), 2.22 (1H, m,
H-7a), 2.09 (3H, s, H-22), 1.81 (1H, m, H-9), 1.04 (3H, s,
H-18), 0.92 (3H, s, H-17), 0.78 (3H, s, H-20). 13C NMR (125
Hz, CD3OD) δ: 171.4 (C-21), 139.3 (C-8), 128.0 (C-14), 73.8
(C-15), 66.6 (C-16), 65.7 (C-19), 64.7 (C-2), 55.1 (C-5), 50.8
(C-9), 48.0 (C-1), 44.4 (C-3), 40.5 (C-4), 39.6 (C-10), 37.5
(C-13), 36.2 (C-7), 31.5 (C-12), 27.3 (C-18) 22.7 (C-17),
22.1 (C-6), 21.0 (C-22), 18.6 (C-11), 16.7 (C-20). Compound
3 was characterized as 16-acetylkirenol by comparison of the
physical and spectral data with literature[5].
Compound 4 Yellow powder, ESI-MS m/z 330 [M + H]
+, 353 [M + Na] +, 329 [M − H]+, C17H14O7. 1H NMR (500
MHz, CDOD) δ: 7.64 (1H, d, J = 2.2 Hz, H-2), 7.54 (1H, dd,
LIU Jia, et al. /Chinese Journal of Natural Medicines 2012, 10(1): 13−15
2012 年 1 月 第 10 卷 第 1 期 Chin J Nat Med Jan. 2012 Vol. 10 No. 1 15

J = 8.5, 2.2 Hz, H-6), 6.90 (1H, d, J = 8.5 Hz, H-5) 6.56 (1H,
d, J = 2.2 Hz, H-8), 6.31 (1H, d, J = 2.2 Hz, H-6), 3.87 (3H, s,
2-OCH3), 3.80 (3H, s, 7-OCH3). 13C NMR (125Hz, CD3OD)
δ: 180.1 (C-4), 167.3 (C-7), 162.8 (C-5), 158.3 (C-9), 150.1
(C-2), 146.5 (C-3), 139.8 (C-3), 122.9 (C-6), 122.4 (C-1),
116.6 (C-5), 116.4 (C-2), 160.7 (C-10), 98.9 (C-6), 93.1
(C-8), 60.5 (3-OCH3), 56.5 (7-OCH3). Compound 4 was
characterized as 3, 7-dimethylquercetin by comparison of the
physical and spectral data with the reported data[6].




Fig. 1 Structure and selected HMBC correlations of com-
pound 1

4 Cytotoxicity Assay
Colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-
tetrazolium bromide (MTT) method[7-8] was used to investi-
gate the cytotoxic activity of compound 1, with cisplatin,
DDP as positive controls (IC50 2.5 μg·mL–1). Compound 1
showed potential activity, with the IC50 of 58 μg·mL-1.
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从腺梗豨莶中分离得到的一种具有细胞毒活性的新氨基甲酸酯
刘 佳 1, 3, 陈 荣 3, 聂 煜 2, 冯 林 3, 李海岛 3, 梁敬钰 1*
1 中国药科大学天然药物化学教研室, 南京 210009；
2 中国药科大学生命科学与技术教研室, 南京 210009；
3 江苏先声药物研究有限公司, 南京 210042
【摘 要】 目的：对腺梗豨莶(Siegesbeckia pubescens)地上部分进行化学成分的研究。方法：运用萃取、结晶及各种柱层析
方法进行分离纯化, 并通过理化鉴别和波谱分析鉴定化合物结构。此外, 测定了化合物 1 对 A549 人肺癌细胞的细胞毒活性。结
果：从腺梗豨莶中分离鉴定了 4 个化合物 , 分别为 (E)-3-(3-oxobut-1-enyl)phenyl dimethylcarbamate (1), ent-2-oxo-15, 16,
19-trihydroxypimar-8(14)-ene (2), 16-acetylkirenol (3) 和 3, 7-dimethylquercetin (4)。结论：化合物 1 为新化合物, MTT 细胞毒活性
显示其 IC50为 58 μg·mL–1。
【关键词】 豨腺梗 莶; 化学成分; 细胞毒

【基金项目】 江苏省自然科学基金 (No. BK2009470)资助项目