A new amide and a new monoterpene from the seeds of Clausena lansium

Article abstract of DOI:10.1080/14786419.2012.678354

A new amide (2) and a new monoterpene (6) were isolated from the seeds of Clausena lansium together with four known ones (1, 3-5). The structures of the new compounds were elucidated by extensive spectroscopic analyses. The absolute configurations of 1, 2 and 6 were determined by optical rotation and the modified Mosher's method.

Full text of DOI:10.1080/14786419.2012.678354

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A new amide and a new monoterpene
from the seeds of Clausena lansium
Guo-Cai Wang a b c , Wen Li a b , Ying Wang a b c , Xiao-Qi Zhang a b
, Yao-Lan Li a b & Wen-Cai Ye a b c
a Institute of Traditional Chinese Medicine and Natural Products,
College of Pharmacy, Jinan University, Guangzhou 510632, P.R.
China
b Guangdong Province Key Laboratory of Pharmacodynamic
Constituents of TCM and New Drugs Research, Jinan University,
Guangzhou 510632, P.R. China
c JNU-HKUST Joint Laboratory for Neuroscience and Innovative
Drug Research, Guangzhou 510632, P.R. China
Version of record first published: 05 Apr 2012.
To cite this article: Guo-Cai Wang, Wen Li, Ying Wang, Xiao-Qi Zhang, Yao-Lan Li & Wen-Cai Ye
(2012): A new amide and a new monoterpene from the seeds of Clausena lansium , Natural Product
Research: Formerly Natural Product Letters, DOI:10.1080/14786419.2012.678354
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Natural Product Research
2012, 1–5, iFirst
A new amide and a new monoterpene from the seeds of Clausena lansium
Guo-Cai Wangabc, Wen Liab, Ying Wangabc, Xiao-Qi Zhangab, Yao-Lan Liab and
Wen-Cai Yeabc
*
aInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy,
Jinan University, Guangzhou 510632, P.R. China; Guangdong Province Key Laboratory of
b
Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou
510632, P.R. China; JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug
Research, Guangzhou 510632, P.R. China
c
(Received 5 September 2011; final version received 2 March 2012)
A new amide (2) and a new monoterpene (6) were isolated from the seeds of
Clausena lansium together with four known ones (1, 3–5). The structures of the
new compounds were elucidated by extensive spectroscopic analyses. The
absolute configurations of 1, 2 and 6 were determined by optical rotation and
the modified Mosher’s method.
Keywords: Clausena lansium; rutaceae; amide; monoterpene
1. Introduction
The plant Clausena lansium, a fruit tree, is widely distributed in the southern area of China
(Huang, 1959). The leaves and roots of the plant are used as folk medicines for the
treatment of coughs, asthma, dermatological disease, viral hepatitis and gastro-intestinal
diseases (Li, James, & Farouk, 1991). The seeds are used to treat acute and chronic gastro-
intestinal inflammation, ulcers, etc. (Lin, 1989). Previous phytochemical investigations on
the plant had led to the isolation of some carbazole alkaloids and amides (Kumar,
Vallipuram, Adebajo, & Reisch, 1995; Li et al., 1991; Lin, 1989; Yang, Chen, & Huang,
1988). In this article, we report a new amide (2) and a new monoterpene (6) from the seeds
of C. lansium together with four known ones (1, 3–5). The structures of the new
compounds were elucidated by extensive spectroscopic analyses. The known compounds
were identified by comparing their physical and spectroscopic data with those reported in
the literatures. The absolute configurations of 1, 2 and 6 were determined by optical
rotation and the modified Mosher’s method.
2. Results and discussion
The air-dried seeds of C. lansium were extracted with 95% EtOH. The extract was
suspended in H2O and successively partitioned with cyclohexane, ethyl acetate and
n-butanol. The EtOAc extract was chromatographed over silica gel, Sephadex LH-20 and
ODS columns to afford compounds 1–6 (Figure 1).
*Corresponding author. Email: chywc@yahoo.com.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.678354
http://www.tandfonline.com

2
G.-C. Wang et al.
O
1
O
1'
OR
4
3
N
R
N
2'
3'
2
CH3
3 R = H
4 R = CH3
1 R = H
2 R = Glc
7
OH
4
1
5
6
O
N
3
2
OH
10
8
CH3
9
5
6
Figure 1. Chemical structures of 1–6.
Compound 2 was isolated as yellow oil. The HR–ESI–MS of 2 showed a quasi-
molecular ion at m/z 466.1842 [M þ Na]þ (calcd for C24H29NO7Na, 466.1836), consistent
with the molecular formula C24H29NO7. The IR spectrum suggested the presence of
hydroxyl group (3407 cmꢀ1) and carbonyl group (1646 cmꢀ1). The 1H-NMR spectrum of 2
displayed two olefinic protons at ꢀH 7.40 (1H, d, J ¼ 15.2 Hz) and 6.98 (1H, d, J ¼ 15.2 Hz),
10 aromatic protons at ꢀH 7.27–7.58 (10H, m), one methyl at ꢀH 3.02 (3H, s) and an
anomeric proton of sugar at ꢀH 4.13 (1H, d, J ¼ 6.3 Hz). A total of 24 carbon signals were
observed in the 13C-NMR and DEPT spectra of 2, including signals of a carbonyl
(ꢀC 166.1), a double bond (ꢀC 139.6, 119.2), a methyl (ꢀC 34.6), a glucose unit (ꢀC 100.4,
73.5, 76.5, 70.1, 76.3, 61.1) and the signals of two benzene rings. The above data were very
similar to those of 1 (Maneerat, Tha-in, Cheenpracha, Prawat, & Laphookhieo, 2011),
except that 2 had an additional glucose unit signals. The HMBC spectrum of 2 showed the
correlation between H-100 (ꢀH 4.13) and C-20 (ꢀC 76.5), indicating the glucose unit was
connected to C-20 position. Hydrolysis of 2 yielded 1 and D-glucose. The NMR data of 1
were consistent with those of Clausenalansamide B (Maneerat et al., 2011), but the
configuration of C-20 was not determined. The optical rotation of 1 was then measured,
and it was negative as that of (R)-aegeline (Ahmed, Ahmad, Mahendra, & Shaheer, 2004),
suggesting the configuration of C-20 in 1 was R. Thus, 2 was elucidated as (20R)-N-
(20phenylethyl)-N-methylcinnamamide 20-O-ꢁ-D-glucopyranoside.
Compound 6 was obtained as white powder. The HR–ESI–MS gave a quasi-molecular
ion at m/z 171.1375 [M þ H]þ (calcd for C10H19O2, 171.1380), suggesting the molecular
1
formula of 6 was C10H18O2. The H-NMR spectrum showed the signals of two olefinic
protons at ꢀH 5.00 (1H, d, J ¼ 0.5 Hz) and 4.91 (1H, d, J ¼ 0.5 Hz), one oxy-methine at
ꢀH 4.43 (1H, m), and an isopropyl group at ꢀH 1.59 (1H, m), 0.91 (3H, d, J ¼ 6.9 Hz) and
0.93 (3H, d, J ¼ 6.9 Hz). The 13C-NMR and DEPT spectra displayed 10 carbon signals
including two olefinic carbons at ꢀC 151.6 and 103.4, a quaternary carbon at ꢀC 75.7, as
well as an isopropyl group at ꢀC 44.5, 16.9 and 16.9. All the above NMR data suggested
that 6 was a menthane-type monoterpene (Ohloff & Uhde, 1965). The positions of the
double bond at C-4 and C-7 and two hydroxyl groups at C-1 and C-3 were elucidated by
1
the H-1H COSY and HMBC spectra. The relative configuration of 6 was elucidated
by the ROESY correlations (Figure 2). The absolute configuration of 6 was confirmed by
application of the modified Mosher’s method (Ohtani, Kusumi, Kashman, & Kakisawa,
1991). Differences of proton chemical shift (Dꢀ values, ꢀS–ꢀR) between (S)-MTPA ester

Natural Product Research
3
+0.01
+0.02
7
OR
-0.01
-0.04
+0.03
+0.05
5
4
6
3
2
OH
1
10
8
-0.04
9
6a: R=(S)-MTPA
6b: R=(R)-MTPA
Figure 2. Key ROESY correlations of 6, and Dꢀ values (ꢀS–ꢀR) of the MTPA esters 6a and 6b.
(6a) and (R)-MTPA ester (6b) (Figure 2) indicated the configuration of C-3 was R.
Hence, 6 was identified as (1R), (3R)-4-methylene-1-(1-methylethyl)-(1,3)-cyclohexanediol.
The other three known compounds were identified as phenethyl cinnamide (3) (Que,
Zhao, & Zhang, 2008), lansiumamide C (4) (Lin, 1989), and lansiumamide I (5) (Bayer &
Maier, 2004), respectively, by a comparison of their physical and spectroscopic data with
those of literatures.
3. Experimental
3.1. General methods
Melting points were determined on an XT-4 micro-melting-point apparatus. Optical
rotations were measured on a JASCO-P-1020 polarimeter. UV spectra were recorded on a
JASCO V-550 UV/VIS spectrophotometer; ꢂmax (log ") in nm. IR spectra (KBr) were
1
measured by a JASCO FT/IR-480 plus spectrometer; in cmꢀ1. H-, 13C- and 2D-NMR
spectra were recorded on a Bruker-AV-400 spectrometer; ꢀ in ppm relative to (CH3)4Si, J
in Hz. HR–ESI–MS spectra were obtained on an Agilent 6210 ESI/TOF mass
spectrometer; in m/z. For column chromatography (CC), silica gel (200–300 mesh,
Qingdao Marine Chemical Factory, Qingdao, P.R. China) and Sephadex LH-20
(Pharmacia) were used. Precoated silica gel GF254 plates (Qingdao Marine Chemical
Factory, Qingdao, P.R. China) were used for TLC.
3.2. Plant material
The seeds of C. lansium were collected in Qingpin TCM Trade Center, Guangzhou,
Guangdong province of China, in July 2008, and authenticated by Professor Zhou Guang-
Xiong of Jinan University. A voucher specimen (no. 080723) was deposited in the Institute
of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou,
P.R. China.
3.3. Extraction and isolation
The seeds of C. lansium (10.0 kg) were extracted with 95% EtOH. The extract was
suspended in H2O and successively partitioned with cyclohexane, ethyl acetate and
n-butanol to afford 125, 135 and 147 g residues, respectively. The EtOAc extract was
subjected to a silica gel column (CHCl3–CH3OH, 100 : 0 ! 0 : 100) to give 10 major
fractions. Fraction 3 was re-chromatographed on a silica gel column (CHCl3–CH3OH,
99 : 1 ! 70 : 30) to afford 1 (10 mg), 3 (21 mg) and 4 (29 mg). Fraction 4 was
re-chromatographed on a silica gel column (CHCl3–CH3OH, 98:2!70:30) to afford 2
(13 mg), 5 (32 mg) and 6 (15 mg).

4
G.-C. Wang et al.
3.3.1. Compound 2
20
Yellow oil, ½ꢃꢁD ¼ ꢀ46ꢂ (c 0.40, CH3OH). UV (MeOH) ꢂmax : 209, 281 nm. IR (KBr) ꢄmax
:
3407, 2924, 1646, 1591, 1404, 1078, 763, 703 cmꢀ1. HR–ESI–MS m/z: 466.1842 [M þ Na]þ
(calcd for C24H29NO7Na, 466.1836). 1H-NMR (DMSO-d6, 400 MHz) ꢀ: 6.98 (1H, d,
J ¼ 15.2 Hz, H-2), 7.40 (1H, d, J ¼ 15.2 Hz, H-3), 3.72 (2H, m, H-10), 5.10 (1H, d,
J ¼ 5.8 Hz, H-20), 7.27–7.58 (10H, m, Ar), 3.02 (3H, s, Me), 4.13 (1H, d, J ¼ 6.3 Hz, H-100),
3.05–3.74 (6H, m, H-200–H-600). 13C-NMR (DMSO-d6, 100 MHz) ꢀ: 166.1 (C-1), 119.2 (C-
2), 139.6 (C-3), 135.1 (C-4), 55.1 (C-10), 76.7 (C-20), 139.0 (C-30), 126.6–128.3 (Ar), 34.6
(Me), 100.4 (C-100), 73.5 (C-200), 76.5 (C-300), 70.1 (C-400), 76.3 (C-500), 61.1 (C-600).
3.3.2. Compound 6
20
White powder, m.p. 75–78ꢂC, ½ꢃꢁD ¼ þ27ꢂ (c 0.50, CH3OH). UV (MeOH) ꢂmax : 240 nm.
IR ꢄmax : 3383, 2983, 2364, 1655, 1396, 1073 cmꢀ1. HR–ESI–MS m/z : 171.1375 [M þ H]þ
1
(calcd for C10H19O2, 171.1380). H-NMR (CDCl3, 400 MHz) ꢀ : 1.31 (1H, m, H-2a), 2.07
(1H, m, H-2b), 4.43 (1H, m, H-3), 2.28 (1H, m, Ha-5), 2.42 (1H, m, Hb-5), 1.43 (1H, m, Ha-
6), 1.64 (1H, m, Hb-6), 5.00 (1H, d, J ¼ 0.5 Hz, Ha-7), 4.91 (1H, d, J ¼ 0.5 Hz, Hb-7), 0.91
(3H, d, J ¼ 6.9 Hz, H-9), 0.93 (3H, d, J ¼ 6.9 Hz, H-10). 13C-NMR (CDCl3, 100 MHz)
ꢀ: 75.7 (C-1), 52.5 (C-2), 69.2 (C-3), 151.6 (C-4), 29.4 (C-5), 35.1 (C-6), 103.4 (C-7), 44.5
(C-8), 16.9 (C-9, C-10).
3.4. Hydrolysis of 2 and determination of the absolute configuration of sugar
Compound 2 (3.0 mg) was dissolved in 1N HCl (2.0 mL) and heated at 80ꢂC for 3 h. The
solution was evaporated with a stream of N2. The reaction mixture was dissolved in H2O
and the aglycone was extracted with CH2Cl2, which was identified as 1. The aqueous layer
was evaporated to give a residue. Then, dry pyridine (1.0 mL) and L-cysteine methyl ester
hydrochloride (3.0 mg) were added to the residue. The mixture was heated at 80ꢂC for 2 h
and then dried by N2. N-(trimethylsilyl) imidazole (0.2 mL) was added, and the mixture
was heated at 80ꢂC for 1 h. Finally, H2O (1.0 mL) was added to stop the reaction, and the
aqueous layer was extracted by cyclohexane (1.0 mL). The organic layer was analysed by
gas chromatography using trimethylsilylated derivatives of standard sugars as references
(Ikeda, Tsumagari, & Nohara, 2000). As a result, D-glucose from the hydrolyzate of 2 was
detected.
3.5. Preparation of MTPA esters of 6
Compound 6 (3.5 mg) was dissolved in 0.5 mL pyridine, and treated with (R)-(ꢀ)-
ꢃ-methoxy-ꢃ-(trifluoromethyl) phenylacetyl chloride (10 mL) for 24 h in an anhydrous
circumstance at room temperature and then dried in vacuum. The reaction mixture was
poured into water (5.0 mL) and extracted with EtOAc (5.0 mL). The EtOAc extract was
purified by silica gel CC [n-hexane-EtOAc (75 : 25)] to yield (S)-MTPA ester (6a, 1.5 mg).
(R)-MTPA ester (6b, 1.4 mg) was obtained using the same method by treatment of 6
(3.7 mg) with (S)-(þ)-ꢃ-methoxy-ꢃ-(trifluoromethyl) phenylacetyl chloride (10 mL).
Acknowledgements
This study was supported by grants from the National Natural Science Foundation (no. 81001374),
Program for Changjiang Scholars and Innovative Research Team in University (IRT0965) and
Research Team Program of Natural Science Foundation of Guangdong Province
(no. 8351063201000003).

Natural Product Research
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