Cytotoxic amides and quinolones from Clausena lansium

Article abstract of DOI:10.1002/hlca.201300323

Two new amides, claulansamides A and B (1 and 2, resp.), together with five known amides, 3-7 and six known quinolones, 8-13, were isolated from the stems and roots of Clausena lansium. Their structures were elucidated on the basis of extensive spectroscopic methods. Their absolute configurations were determined by single-crystal X-ray diffraction technique, CD, and optical rotation. HPLC Chiral separation of 1 afforded two enantiomers, (+)- and (-)-claulansamide A, respectively. Compounds 9, 12, and 13 were isolated from the genus Clausena for the first time. All compounds were evaluated for their cytotoxic activities against A549, BGC-823, and HeLa cancer cell lines. However, only 9 showed cytotoxic activity against A549 cell line with an IC₅₀ value of 46.3 μM, and 11 against BGC-823 and HeLa cell lines with the IC₅₀ values of 55.0 and 14.7 μM, respectively. Copyright

Full text of DOI:10.1002/hlca.201300323

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Helvetica Chimica Acta – Vol. 97 (2014)
Cytotoxic Amides and Quinolones from Clausena lansium
by Wei-Wu Songa)b), Guang-Zhi Zenga), Wen-Wen Penga), Ke-Xian Chena)b), and Ning-Hua Tan*a)
a) State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650201, P. R. China
(phone: þ 86-871-65223800; fax: þ 86-871-65223800; e-mail: nhtan@mail.kib.ac.cn)
b) University of Chinese Academy of Sciences, Beijing 100049, P. R. China
Two new amides, claulansamides A and B (1 and 2, resp.), together with five known amides, 3 – 7 and
six known quinolones, 8 – 13, were isolated from the stems and roots of Clausena lansium. Their
structures were elucidated on the basis of extensive spectroscopic methods. Their absolute configurations
were determined by single-crystal X-ray diffraction technique, CD, and optical rotation. HPLC Chiral
separation of 1 afforded two enantiomers, (þ)- and (ꢀ)-claulansamide A, respectively. Compounds 9, 12,
and 13 were isolated from the genus Clausena for the first time. All compounds were evaluated for their
cytotoxic activities against A549, BGC-823, and HeLa cancer cell lines. However, only 9 showed
cytotoxic activity against A549 cell line with an IC50 value of 46.3 mm, and 11 against BGC-823 and HeLa
cell lines with the IC50 values of 55.0 and 14.7 mm, respectively.
Introduction. – Clausena lansium (Lour.) Skeels (Rutaceae) is a plant widely
distributed in South China. Its fruits have been used as a folk medicine to treat
indigestion, cold, cough, and stomach pain [1]; its leaves and roots for the treatment of
cough, asthma, dermatological diseases, viral hepatitis, and gastro-intestinal disease;
and its seeds for treating acute and chronic gastrointestinal inflammation, and ulcer [2].
Previous studies revealed that the genus Clausena contains mainly coumarins, cabazole
alkaloids, amides, and terpenoids [3 – 7]. Huang and co-workers isolated the first
amide, named clausenamide, featuring reduction of SGPT (serum glutamic-pyruvic
transaminase) activity from the leaves of C. lansium [8 – 10]. So far, ca. 30 amides have
been isolated from this plant [11 – 17]. Some of them showed cytotoxic and
hepatoprotective activities [13][14]. There are also about ten quinolones isolated
from this plant [4][18 – 22]. Our efforts to obtain bioactive constituents from C. lansium
has led to the isolation of two new amides, claulansamides A and B (1 and 2, resp.),
together with five known amides, 3 – 7, and six known quinolones, 8 – 13. Herein, we
report the isolation, structure elucidation, and cytotoxicity of these compounds.
Results and Discussion. – Claulansamide A (1) was obtained as colorless crystals.
The HR-EI-MS showed a molecular-ion peak at m/z 297.1364 (Mþ, C18H19NO3þ ; calc.
297.1365), suggesting ten degrees of unsaturation. The UV spectrum showed typical
maximum absorptions of a conjugated benzene chromophore at lmax 208 and 256 nm.
The IR spectrum displayed absorption bands of OH (3423 cmꢀ1) and amide CO
1
(1640 cmꢀ1) groups. The H-NMR spectrum (Table) revealed the presence of two
benzene rings (d(H) 7.20 – 7.37 (10 H, overlapped)), three O-bearing CH groups (d(H)
ꢀ 2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

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5.14 (dd, J ¼ 8.8, 3.2), 4.98 (d, J ¼ 9.4), and 4.15 (d, J ¼ 9.4)), one CH2 group (d(H) 3.29
(dd, J ¼ 14.4, 8.8) and 3.13 (dd, J ¼ 14.4, 3.2), and one MeN group (d(H) 3.02 (s)). The
13C-NMR spectrum (Table) exhibited 18 signals attributable to one CO group (d(C)
170.0), twelve benzene C-atoms (d(C) 137.8 – 126.6), three O-bearing CH groups (d(C)
88.7, 73.7, and 70.2), one CH2 group (d(C) 37.8), and one MeN C-atom (d(C) 31.3).
These data evidenced the presence of two benzene rings and a pyran ring bearing a CO
group. The latter was supported by the HMBCs from HꢀC(7) to C(9) and C(11), from
HꢀC(8) to C(9), from MeN to C(9) and C(11), and from HꢀC(11) to C(9), and by the
1H,1H-COSY correlations between HꢀC(7) and HꢀC(8), and between HꢀC(11) and
HꢀC(12). Positions of the CH2 group and two benzene rings were established by the
HMBC cross-peaks from HꢀC(8) and HꢀC(7) to C(1), and from HꢀC(12) to C(11)
and C(13). These analyses suggested that 1 was an amide, and its constitution was
established (Fig. 1).
Fig. 1. Structures of compounds 1 and 2
The relative configuration of 1 was determined by the 1H-NMR coupling constants
1
and ROESY spectrum. In the H-NMR spectrum, the coupling J(7,8) constant is
9.4 Hz, and there is a correlation between HꢀC(7) and HꢀC(12) in the ROESY
spectrum. These data indicated that HꢀC(7) and HꢀC(12) are in the same orientation,
but HꢀC(8) and Hꢀ(11) are in another orientation (Fig. 2). To establish the
configuration, 1 was crystallized from acetone to afford a crystal of the monoclinic
space group P21/c, which was analyzed by X-ray crystallography with CuKa (l ¼
1.54178 ꢂ) radiation (Fig. 3). Finally, 1 was determined as a racemate by single-crystal
X-ray diffraction analysis. Subsequent HPLC separation of 1 on chiral column gave two
individual enantiomers (ꢀ)-1 and (þ)-1, which displayed opposite Cotton effects in
their electronic CD (ECD) spectra and opposite optical rotations. The HPLC integral
area of the two enantiomers, (ꢀ)-1 and (þ)-1), were in a ratio of ca. 1:2. Furthermore,
the geometry was optimized using DFT at the B3LYP/CC-pVDZ level on the basis of
the crystal structure. The harmonic vibrational frequency was then calculated to
confirm its stability at the same level. The ECD spectrum and the optical rotation

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Table. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; CDCl3) of Compounds 1 and 2. d in ppm, J in
Hz.
Position
1
2
d(H)
d(C)
d(H)
d(C)
1
2,6
3,5
4
7
8
9
11
12
137.8
126.6
128.5
128.5
73.7
70.2
170.0
88.7
137.6
126.6
128.5
128.5
78.0
70.2
171.1
89.0
7.37 (overlapped)
7.34 (overlapped)
7.34 (overlapped)
4.98 (d, J ¼ 9.4)
4.15 (d, J ¼ 9.4)
7.40 (overlapped)
7.40 (overlapped)
7.36 (overlapped)
4.51 (d, J ¼ 9.6)
3.77 (d, J ¼ 9.6)
5.14 (dd, J ¼ 8.8, 3.2)
3.29 (dd, J ¼ 14.4, 8.8),
3.13 (dd, J ¼ 14.4, 3.2)
5.17 (d, J ¼ 2.6)
37.8
3.23 (dd, J ¼ 14.4, 2.6),
3.07 (overlapped)
40.0
13
135.5
129.3
128.7
127.1
31.3
134.5
130.1
128.5
127.1
29.7
14,18
15, 17
16
7.20 (overlapped)
7.29 (overlapped)
7.26 (overlapped)
3.02 (s)
7.30 (overlapped)
7.31 (overlapped)
7.27 (overlapped)
3.03 (s)
MeN
(589.3 nm) of the optimized geometry were calculated at the B3LYP/Aug-CC-pVDZ
level in MeCN [23]. All the calculations were carried out with the Gaussian 03
program. The computed and experimental ECD spectra are shown together in Fig. 4.
The calculated optical rotation value of (þ)-1 was þ 28.39 (experimental value: þ
24.0) and of (ꢀ)-1 was ꢀ 28.39 (experimental value: ꢀ 36.3). The experimental
optical rotation value of 1, þ 24.8, indicated that 1 was a mixture (ꢀ)-1/(þ)-1 with a
ratio of ca. 1:2. Accordingly, the absolute configurations of (þ)-1 and (ꢀ)-1 were
established.
Fig. 2. Key HMBC (H ! C) and ROESY (H $ H)
correlations of compound 1
Claulansamide B (2) has the same molecular formula as 1 determined by HR-EI-
MS (m/z 297.1362 (Mþ, C18H19NO3þ, calc. 297.1365)). Comparison of the NMR data of 2
with those of 1 revealed that they have the same planar structure, and the only
difference was the configuration at C(11). In the ROESY spectrum of 2, the
correlation between HꢀC(7) and HꢀC(11) indicated that HꢀC(7) and HꢀC(11) are in
the same orientation. Comparison of the experimental optical rotation ([a]2D4:0 ¼ þ1.9
(c ¼ 0.35, MeOH)) with the calculated value of ꢁ 54.9 indicated that 2 was a mixture
with a ratio of ca. to 1:1.

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301
Fig. 3. X-Ray crystal structures of compound 1
Fig. 4. Calculated ECD spectrum of (þ)-1 in
MeCN (—); experimental ECD spectrum of (þ)-
1 in MeCN ( ···· ); and experimental ECD
spectrum of (ꢀ)-1 in MeCN (– – –)
The known compounds (Fig. 5) were identified as clausenalansamide A (3) [13],
clausenalansamide B (4) [13][15], secodemethylclausenamide (5) [16], (ꢀ)-(R)-
tembamide (6) [17], (ꢀ)-clausenamide (7) [9][10], 4-methoxy-1-methyl-2-quinolone
(8) [20], 6-hydroxy-4-methoxy-1-methyl-2-quinolone (9) [24], 4-methoxy-1H-2-quino-
lone (10) [21], dictamine (11) [22], atanine (12) [25], and (þ)-(S)-platydesmine (13)
[26] by comparing the NMR and MS data with those in the literature. The absolute
configurations of 3 and 5 were determined by induced CD experiments [27], and of 4,
6, 7, and 13 were determined by comparing the optical rotation data with those in the
literature.
Cytotoxic activities of all compounds were evaluated in vitro against A549 (human
lung adenocarcinoma), BGC-823 (human gastric cancer), and HeLa (cervical cancer)
cancer cell lines. Only 9 showed cytotoxic activitiy against A549 cancer cell line with an
IC50 value of 46.3 mm, and 11 against BGC-823 and HeLa cancer cell lines with the IC50
values of 55.0 and 14.7 mm, respectively.
This work was supported by the National New Drug Innovation Major Project of China
(2011ZX09307-002-02), the National Natural Science Foundation of China (U1032602, 91013002, and

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Fig. 5. Structures of compounds 3 – 13
30725048), the National Basic Research Program of China (2009CB522300), the Fund of Chinese
Academy of Sciences (Hundred Talents Program), and the Natural Science Foundation of Yunnan
Province (2012GA003). The authors are grateful to the members of the analytical group of the State Key
Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences, for single-crystal X-ray diffraction analysis, for determining the optical rotations,
and for recording the CD, IR, UV, NMR, and mass spectra.
Experimental Part
General. TLC: Silica gel GF254 plates (Qingdao Yuminyuan Silica Reagent Factory, Qingdao, P. R.
China); visualization with UV light (254 and 365 nm), and by spraying with 5% aq. H2SO4 soln., followed
by heating. Column chromatography (CC): silica gel (SiO2; 200 – 300 mesh; Qingdao Yuminyuan Silica
Reagent Factory, Qingdao, P. R. China); Sephadex LH-20 (Pharmacia, Amersham Biosciences, SE-
Uppsala); RP-18 silical gel (40 – 60 mm, Merck, DE-Darmstadt); and MCI gel (CHP-20P, 70 – 150 mm,
Mitsubishi Chemical Corporation, Japan). MPLC: EZ Purifier 100/200 equipped with a UV detector
(Lisure Science (Suzhou) Co., Ltd., P. R. China) and a RP-18 column (3.5 ꢂ 25 or 4.5 ꢂ 40 cm, 40 –
60 mm) or a MCI column (4.5 ꢂ 40 cm); flow rate, 25 ml/min. Semiprep. HPLC: Agilent 1100 apparatus
equipped with a UV detector and CHIRALCELꢀ OD-H (10 ꢂ 250 mm, 5 mm, Daicel Chiral
Technologies (China) Co., Ltd.); flow rate, 2 ml/min. Prep. reversed-phase (RP) HPLC: Agilent 1100
apparatus equipped with a UV detector and a SunFire OBD (1.9 ꢂ 25 cm, 5 mm, Waters) column, flow
rate, 10 ml/min. M.p.: Gongyi Yuhua X-4 digital melting-point apparatus. Optical rotations: Jasco P-1020
Polarimeter. UV Spectra: Shimadzu UV-2401PC spectrophotometer; in nm. CD Spectra: Agilent Applied
Photophysics spectrometer; in nm. IR Spectra: Bruker Tensor 27 FT-IR spectrometer; KBr pellets; in
1
cmꢀ1. H- and 13C-NMR spectra: Bruker AM-400 (at 400 and 100 MHz, resp.), DRX-500 (at 500 and
125 MHz, resp.), or Bruker AV-600 (at 600 and 150 MHz), Spectrometer in (D6)acetone, CDCl3, or
(D5)pyridine at r.t.; d in ppm rel. to TMS, J in Hz. ESI-MS: Waters Xevo TQ-S or Bruker HCT/Esquire;

Helvetica Chimica Acta – Vol. 97 (2014)
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in m/z. EI-MS and HR-EI-MS: Waters AutoSpec Premier P776; in m/z. X-Ray crystallography: Bruker
APEX DUO diffractometer.
Plant Material. The stems and roots of C. lansium were collected in Hekou, Yunnan Province, P. R.
China, in September 2010, and identified by Prof. Yu-Min Shui, Kunming Institute of Botany, Chinese
Academy of Sciences. Avoucher specimen (No. 0599043) was deposited with the Herbarium of Kunming
Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation. Air-dried and powdered stems and roots of C. lansium (27 kg) were
extracted and refluxed with MeOH for three times each for 4 h (MeOH, 3 ꢂ 50 l). The extract was
evaporated under reduced pressure to yield a dark brown residue (900 g). The residue was suspended in
MeOH/H2O 7:3 (3 l) and then partitioned with AcOEt (3 ꢂ 2 l). After removing solvent, the AcOEt-
soluble part (406 g) was fractionated by CC (SiO2 (200 – 300 mesh); CHCl3/MeOH 30 :1 to 4 :1) to afford
six fractions, Frs. 1 – 6, on the basis of TLC analysis. Fr. 2 (38 g) was subjected successively to MPLC (RP-
18; MeOH/H2O 10 :90 to 60 :40), CC (SiO2; PE/AcOEt 9 :1 to 7:3), and prep. HPLC (MeCN/H2O
60 :40) to afford 11 (24 mg). Fr. 4 (17.9 g) was separated by MPLC (MCI, MeOH/H2O 10 :90 to 40 :60),
repeated CC (SiO2; PE/acetone 9 :1), and prep. HPLC (MeCN/H2O 60 :40) to give 12 (6 mg). Fr. 5
(6.9 g) was first submitted to MPLC (MCI; MeOH/H2O 5 :95 to 60 :40) and then subjected to CC (SiO2;
PE/acetone 9 :1). Finally, 6 (56 mg), 10 (55 mg), and 13 (152 mg) were obtained by prep. HPLC (MeCN/
H2O 50 :50), (MeCN/H2O 38 :62), and (MeCN/H2O 45 :55), resp. Fr. 6 (77 g) was purified by CC (SiO2;
CHCl3/acetone 15 :1 to 7:3) to afford Fr. 6.1 – Fr. 6.4. Fr. 6.2 (14.3 g) was subjected successively to CC
(SiO2; PE/acetone 5 :1), MPLC (MCI, MeOH/H2O 10 :90 to 60 :40), and MPLC (RP-18; MeOH/H2O
5 :95 to 70 :30) to remove pigments. Then, the residue was further purified by repeated CC (SiO2; CHCl3/
acetone 20 :1; SiO2; CHCl3/AcOEt 20 :1; and SiO2; PE/acetone 9 :1). In the next step by prep. HPLC
(MeCN/H2O 35 :65), six compounds were obtained: 8 (411 mg), 1 (14 mg), 2 (5 mg), 3 (146 mg), 4
(10 mg), and 5 (57 mg). Compound 1 was subjected to HPLC (hexane/iPrOH 20 :80) with a semi-prep.
column on chiral stationary phase and afforded two enantiomers (ꢀ)-1 (5.1 mg) and (þ)-1 (8.4 mg).
Fr. 6.4 (3.8 g) was subjected to CC for three times (Sephadex LH-20; CHCl3/MeOH 1.5 :1, 1:1 or 1:1.5)
and then subjected to MPLC (RP-18; MeOH/H2O 10 :90 to 100 :0). The residue was subjected to
repeated CC (SiO2; PE/acetone 9 :1; SiO2; PE/iPrOH 20 :1, resp.), followed by prep. HPLC (MeCN/H2O
35 :65) to afford 7 (25 mg) and 9 (6 mg).
Claulansamide A (¼2-Benzyl-5-hydroxy-3-methyl-6-phenyl-1,3-oxazinan-4-one; 1): Colorless crys-
tals (acetone). [a]2D3:7 ¼ þ24.8 (c ¼ 1.19, MeCN). M.p. 178 – 1808. UV (MeOH): 208 (4.73), 256 (3.19). IR
1
(KBr): 3423, 2923, 1640, 1453, 1409, 1116, 1080, 1029. H- and 13C-NMR: see the Table. ESI-MS: 298
([M þ H]þ), 320 ([M þ Na]þ), 617 ([2M þ Na]þ). HR-EI-MS: 297.1364 (Mþ, C18H19NOþ3 ; calc.
297.1365).
(þ)-Claulansamide A (¼(2R,5R,6R)-2-Benzyl-5-hydroxy-3-methyl-6-phenyl-1,3-oxazinan-4-one;
(þ)-1). Colorless oil. [a]2D4:6 ¼ þ24.0 (c ¼ 0.14, MeCN). CD (MeCN): 229 (De ꢀ2.79). ESI-MS: 298
([M þ H]þ), 320 ([M þ Na]þ), 617 ([2 M þ Na]þ).
(ꢀ)-Claulansamide A (¼(2S,5S,6S)-2-Benzyl-5-hydroxy-3-methyl-6-phenyl-1,3-oxazinan-4-one; (ꢀ)-
1). Colorless oil. [a]2D4:6 ¼ ꢀ36.3 (c ¼ 0.30, MeCN). CD (MeCN): 231 (De þ 2.45). ESI-MS: 298 ([M þ
H]þ), 320 ([M þ Na]þ), 617 ([2M þ Na]þ).
Claulansamide B (¼(2S,5R,6R)- or (2R,5S,6S)-2-Benzyl-5-hydroxy-3-methyl-6-phenyl-1,3-oxazin-
an-4-one; 2). Colorless oil. [a]2D4:0 ¼ þ1.9 (c ¼ 0.35, MeOH). UV (MeOH): 208 (4.32), 251 (2.90). IR
(KBr): 3427, 2924, 1642, 1495, 1454, 1405, 1136, 1072, 1030. 1H- and 13C-NMR: see the Table. ESI-MS: 298
([M þ H]þ), 320 ([M þ Na]þ), 617 ([2M þ Na]þ). HR-EI-MS: 297.1362 (Mþ, C18H19NOþ3 ; calc.
297.1365).
X-Ray-Crystallographic Analysis of 11). Colorless crystals of 1 were obtained from acetone.
Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX
II CCD, using CuKa radiation. Cell refinement and data reduction were performed with Bruker SAINT.
The structures were solved by direct methods using SHELXS-97. Refinements were performed with
1
)
CCDC-944736 contains supplementary crystallographic data for this article. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.

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Helvetica Chimica Acta – Vol. 97 (2014)
SHELXL-97, using full-matrix least-squares, with anisotropic displacement parameters for all the non-H-
atoms. The H-atoms were placed in calculated positions and refined using a riding model. Molecular
graphics were computed with SHELXP-97.
Crystallographic Data of Claulansamide A (1): C18H19NO3, Mr 297.34, monoclinic, crystal size: 0.38 ꢂ
0.24 ꢂ 0.12 mm. a ¼ 9.7666(2) ꢂ, b ¼ 14.3759(3) ꢂ, c ¼ 10.9139(2) ꢂ, a ¼ 90.008, b ¼ 94.2170(10)8, g ¼
90.008, V¼ 1528.20(5) ꢂ3, T¼ 100(2) K, space group P21/c, Z ¼ 4, m(CuKa) ¼ 0.711 mmꢀ1, 11833
reflections measured, 2691 independent reflections (Rint ¼ 0.0466). The final R1 values were 0.0465
(I > 2s(I)). The final wR(F2) values were 0.1232 (I > 2s(I)). The final R1 value was 0.0472 (all data). The
final wR(F2) value was 0.1240 (all data). The goodness-of-fit on F2 was 1.041.
Cytotoxicity Assay. The cytotoxic activities of all compounds were evaluated against A549, HeLa,
and BGC-823 cancer cell lines using the SRB (sulforhodamine B) assay [28]. The cells were cultured in
RPMI 1640 medium (Sigma). Aliquots of 90 ml were seeded in 96-well flat-bottomed microtiter plates
for 24 h and then treated with serial dilutions of the tested compounds with the maximum concentration
of 20 mg/ml. Each compound was initially dissolved in DMSO and further diluted by the medium to
produce different concentrations. After incubation at 378 and 5% CO2 for 48 h, cells were fixed with 25 ml
of ice-cold 50% CCl3COOH and incubated at 48 for 1 h. After washing, air-drying, and staining for
15 min with 100 ml 0.4% SRB in 1% glacial AcOH, excessive dye was removed by washing with 1%
glacial AcOH. After air-drying the plates, SRB was resuspended in 100 ml 10 mm Tris buffer, and the
absorbance was measured at 560 nm with a plate reader (Molecular Devices, SPECTRA MAX 340). All
tests were performed in triplicate, and results are expressed as IC50 values.
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Received August 26, 2013