Nine 2-(2-phenylethyl)chromone derivatives from the resinous wood of Aquilaria sinensis and their inhibition of LPS-induced NO production in RAW 264.7 cells

Article abstract of DOI:10.1002/ejoc.201200725

A phytochemical investigation of aquilariae lignum resinatum, the resinous wood of Aquilaria sinensis (Lour.) Gilg, led to the isolation of nine new 2-(2-phenylethyl)chromone derivatives, aquilarones A-I (1-9), together with two known analogues (10 and 11

Full text of DOI:10.1002/ejoc.201200725

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FULL PAPER
DOI: 10.1002/ejoc.201200725
Nine 2-(2-Phenylethyl)chromone Derivatives from the Resinous Wood of
Aquilaria sinensis and Their Inhibition of LPS-Induced NO Production in
RAW 264.7 Cells
Dong Chen,[a] Zhengren Xu,[a] Xingyun Chai,[b] Kewu Zeng,[a] Yanxing Jia,[a] Dan Bi,[a]
Zhizhong Ma,[a] and Pengfei Tu*[a]
Keywords: Biological activity / Chromones / Configuration determination / Anti-inflammatory agents / Eaglewood / Natu-
ral products
A phytochemical investigation of aquilariae lignum res-
inatum, the resinous wood of Aquilaria sinensis (Lour.) Gilg,
led to the isolation of nine new 2-(2-phenylethyl)chromone
derivatives, aquilarones A–I (1–9), together with two known
analogues (10 and 11). Their structures were elucidated by
extensive spectroscopic analyses, including UV, IR, NMR,
and electronic circular dichroism (ECD) data, as well as by
chemical methods. The absolute configuration of aquila-
rone A (1) was confirmed by single-crystal X-ray diffraction
analysis of its acetonide derivative 1a. All the compounds ex-
hibited significant inhibition of the nitric oxide production in
RAW 264.7 cells, with IC50 values in the range of 5.12–
22.26 μM. In addition, an HPLC–UV comparison analysis of
the resinous wood and resin-free wood is also included.
necessary and very important for the identification and
quality evaluation of eaglewood.
Introduction
Eaglewood, Chinese name “Chenxiang”, termed aquilar-
iae lignum resinatum (ALR) is derived from the diseased
timber of the Aquilaria species of the family Thymelaeaceae.
It is also called gaharu, jinko, or agarwood. Eaglewood has
been used as incense, perfume, and traditional medicine in
Asian countries. ALR, uniquely originating from the resin-
ous wood of Aquilaria sinensis (Lour.) Gilg, is used as a
sedative, analgesic, and digestive in traditional Chinese
medicine.[1]
As well as being one of the most famous incense and
Chinese medicinal materials, eaglewood is also used as the
timber for expensive fine crafts and decorations all over the
world. Currently, the price of high-quality eaglewood mate-
rial reaches up to one hundred thousand U.S. dollars per
kilogram.[2] However, lots of adulterated and inferior eagle-
wood, which is obtained by injecting perfume into the stems
of A. sinensis or other resin-free woods, is traded in China
and other Southeast Asian markets. Therefore, a simple and
effective method, based on its characteristic constituents, is
Previous studies on eaglewood revealed the presence of
its major components, which are sesquiterpenes and chro-
mone derivatives,[3,4] and it exhibited biological activity as
a laxative, sedative, antimicrobial, antitumor agent, antioxi-
dant, anti-inflammatory activity, and anti-allergen.[5–12]
2-(2-Phenylethyl)chromones, one of the characteristic
constituents of eaglewood, have been discovered only in
Aquilaria plants so far. To the best of our knowledge, since
the first isolation and characterization of 2-(2-phenylethyl)-
chromones from A. agallocha in 1978,[13] only 57 congeners
have been reported.[13–35] Also, little effort has been made to
evaluate their biological activity, except for previous papers
regarding cytotoxicity,[34] neuroprotective evaluation,[11,35]
as well as a preliminary test of the anti-inflammatory ac-
tivity of the EtOH extract of A. sinensis leaves.[8]
We systematically investigated the resinous wood of A.
sinensis in order to find new and bioactive constituents and
to provide chemical markers for its quality control. This led
to the isolation and structural elucidation of nine new 2-(2-
phenylethyl)chromones, namely, aquilarones A–I (1–9),
along with two known ones (10 and 11) (Figure 1).[35] Their
structures were elucidated by extensive UV, IR, NMR, and
electronic circular dichroism (ECD) spectroscopic analysis
as well as by chemical methods. Compounds 1–11 exhibited
significant inhibition in an anti-inflammatory assay against
nitric oxide (NO) production in RAW 264.7 cells. We de-
scribe herein the isolation, structural elucidation, and the
anti-inflammation evaluation of these isolates.
[a] State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences,
Peking University Health Science Center,
No. 38 Xueyuan Road, Haidian District, Beijing 100191, China
Fax: +86-10-8280-2750
E-mail: pengfeitu@vip.163.com
[b] Modern Research Center for Traditional Chinese Medicine,
Beijing University of Chinese Medicine,
Beijing 100029, China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200725.
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D. Chen, Z. Xu, X. Chai, K. Zeng, Y. Jia, D. Bi, Z. Ma, P. Tu
FULL PAPER
Figure 1. Structures of aquilarones A–I (1–9) and two congeners (10, 11).
The relative stereochemistry of 1 was determined by
Results and Discussion
analysis of the NOESY spectrum, in which a cross-peak of
Aquilarone A (1) was obtained as a white amorphous pow- 5-H/7-H was observed (see Supporting Information), infer-
der. Its HRMS (ESI) gave an [M + H]+ ion peak at m/z = ring their cofacial assignment. However, the absolute con-
365.1230 (calcd. 365.1240), which corresponds to the mo- figuration was resolved only by a combination of CD spec-
lecular formula C18H20O8. The IR absorptions suggested troscopy (exciton chirality method) of its 5,6-di-p-methoxy-
the presence of hydroxy groups (3359 cm–1) and olefins benzoate derivative and X-ray diffraction analysis of the
1
(1656, 1589 cm–1) in the molecule. The H and 13C NMR acetonide derivative of 1, 1a. The synthetic route for the
spectroscopic data (Tables 1 and 2) revealed the presence of derivatives of 1, 1a–1d, is depicted in Figure 3. Firstly, pro-
a methoxy group, four consecutive methane groups [δH
=
tection of the 1,2-diols with 2,2-dimethoxypropane in the
4.59/δC = 64.0 (C-5), δH = 4.36/δC = 70.0 (C-8), δH = 3.76/ presence of a catalytic amount of pyridinium p-toluenesul-
δC = 66.6 (C-6), and δH = 3.78/δC = 74.5(C-7) ppm], four fonate (PPTS) afforded the acetonide derivative 1a, which
hydroxy groups [δH = 6.07 (8-OH), 5.35 (7-OH), 5.05 (5- confirmed the cis arrangement of 5-OH and 6-OH. Then,
OH), and 4.90 (6-OH) ppm], two methylene groups [δH
=
acetylation of the free hydroxy groups and the phenol group
2.75 (m)/δC = 31.2 (C-7Ј), δH = 2.82 (m)/δC = 34.3 (C-8Ј) with acetic anhydride produced the fully protected deriva-
ppm], and a characteristic ABX coupling system [δH = 6.80 tive 1b, which upon treatment with trifluoroacetic acid
(d)/δC = 112.2 (C-5Ј), δH = 6.66 (d)/δC = 115.6 (C-2Ј), δH
=
(TFA) afforded the 1,2-diol derivative 1c. Finally, esterifica-
6.59/δC = 118.7 (C-6Ј) ppm]. Together, the ABX system, tion with p-methoxybenzoyl chloride yielded the desired
two methylene groups, and the methoxy group construct a 5,6-di-p-methoxybenzoate derivative 1d.
partial (3Ј-hydroxy-4Ј-methoxyphenyl)ethyl structure. This
is supported by HMBC spectroscopy (Figure 2), which cor- effect at 266 nm (benzoate groups) due to a πǞπ* intramo-
relates the methoxy protons to C-4Ј, and both 3Ј-OH (δH lecular charge-transfer transition was observed, which indi-
In the CD spectrum of 1d, a significant positive Cotton
=
8.82 ppm) and 5Ј-H to C-3Ј. The establishment of consecu- cated the clockwise arrangement of the two p-methoxyben-
1
tive methane groups was achieved from the H–1H COSY zoate groups as shown in 1d (Figure 3), thus allowing the
spectrum, which shows cross-peaks for 5-H through 8-H. assignment of the absolute configuration of
1 as
Besides the presence of a carbonyl group (δC = 178.4 ppm, (5S,6S,7S,8R) (Figure 4).[37] Fortunately, a single-crystal of
C-4), three sp2 quaternary carbon atoms [of which two are 1a suitable for an X-ray diffraction experiment on a copper
oxygenated (δC = 146.3, 146.1 ppm)] were observed in the target [with its Flack coefficient of 0.0(2)] was obtained and
13C NMR spectrum. This combined with analysis of the provided unambiguous evidence for the same configuration
remaining key HMBC correlations (Figure 2) from 2Ј-H assignments (Figure 4).[38] Consequently, the structure of 1
and 6Ј-H to C-8Ј, from 7Ј-H and 8Ј-H to C-2, from 8-OH was elucidated as (5S,6S,7S,8R)-2-[2-(3Ј-hydroxy-4Ј-meth-
to C-9, from 5-H to C-9 and C-4, and from 3-H to C-10 oxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetra-
and C-7Ј established its planar structure as 5,6,7,8-tetrahy- hydrochromone.
droxy-2-(3Ј-hydroxy-4Ј-methoxyphenylethyl)-5,6,7,8-tetra-
hydrochromone. It is a 2-(2-phenylethyl)chromone deriva- ous powder. Its molecular formula C17H18O6 was deter-
Aquilarone B (2) was obtained as a pale yellow amorph-
1
tive similar to agarotetrol and isoagarotetrol.[13,16]
mined from the HRMS (ESI) data. The H NMR spectro-
2
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Nine 2-(2-Phenylethyl)chromone Derivatives from Aquilaria sinensis
1
Table 1. H NMR spectroscopic data (in [D6]dimethyl sulfoxide, DMSO) of compounds 1–6,[a] δ in ppm (J in Hz).
1
2
3
4
5
6
3-H
5-H
6.12 s
4.59 dd (5.5, 5.0)
6.13 s
4.58 dd (5.2, 5.0)
5.04 d (5.2)
6.12 s
4.58 dd (5.5, 5.0)
5.09 d (5.5)
6.15 s
4.47 dd (7.0, 3.0)
5.14 d (3.0)
6.08 s
4.47 dd (5.0, 4.0)
5.18 d (5.0)
6.05 s
4.47 dd, (4.5, 4.0)
5.17 d (4.5)
5-OH 5.05 d (5.5)
6-H
6-OH 4.90 d (6.0)
7-H 3.78 ddd (5.0, 4.8, 2.0) 3.78 ddd (4.8, 4.0, 2.0) 3.78 ddd (5.0, 4.8, 2.0) 3.43 ddd (7.0, 4.5, 4.5) 3.84 ddd (7.5, 6.0, 2.0) 3.83 ddd (7.5, 6.0, 2.0)
7-OH 5.35 d (5.0)
3.76 ddd (6.0, 5.0, 2.0) 3.76 ddd (5.6, 5.0, 2.0) 3.76 ddd (6.0, 5.0, 2.0) 3.49 ddd (7.0, 4.5, 4.5) 3.74 ddd (4.0, 4.0, 2.0) 3.74 ddd (4.0, 4.0, 2.0)
4.90 d (5.6) 5.09 d (6.0) 5.34 d (4.5) 4.96 d (4.0) 4.96 d (4.0)
5.35 d (4.8)
4.36 dd (6.0, 4.0)
6.00 d (6.0)
–
5.30 d (5.0)
4.36 dd (6.5, 4.8)
5.90 d (6.5)
7.15 d (8.4.)
6.83 d (8.4)
–
6.83 d (8.4)
7.15 d (8.4)
2.75 m
5.21 d (4.5)
4.32 dd (7.0, 6.5)
5.83 d (6.5)
6.66 d (2.0)
–
5.08 d (6.0)
4.32 dd (7.5, 6.5)
5.80 d (6.5)
6.68 d (2.0)
–
5.10 d (6.0)
4.31 dd (7.5, 6.5)
5.81 d (6.5)
7.01 d (8.0)
6.66 d (8.0)
–
6.66 d (8.0)
7.01 d (8.0)
2.77 m
8-H
4.36 dd (6.5, 4.8)
8-OH 6.07 d (6.5)
2Ј-H
3Ј-H
4Ј-H
5Ј-H
6Ј-H
7Ј-H
8Ј-H
6.66 d (2.0)
–
–
6.80 d (8.0)
6.59 dd (8.0, 2.0)
2.75 m
–
7.16–7.26 (m, 5 H)
–
–
2.75 m
2.82 m
–
–
6.81 d (8.0)
6.61 dd (8.0, 2.0)
2.76 m
6.81 d (8.5)
6.61 dd (2.0, 8.5)
2.76 m
2.82 m
2.82 m
2.83 m
2.83 m
2.83 m
OCH3 3.71 s
OH 8.82 br. s
–
–
3.70 s
–
3.72 s
8.84 br. s
3.72 s
8.82 br. s
–
–
[a] Compounds 2 and 3 measured at 400 MHz, others at 500 MHz.
Table 2. 13C NMR spectroscopic data (in [D6]DMSO) for com- rotation value showed that 2 has the same relative and abso-
pounds 1–6, δ in ppm.[a]
lute configurations of all four of its hydroxy groups as those
No.
1
2
3
4
5
5[b]
6
of 1. Thus, 2 was identified as (5S,6S,7S,8R)-2-(2-phenyl-
ethyl)-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone.
Analysis of the HRMS (ESI) data of aquilarone C (3)
gave a molecular formula of C18H20O7. Comparison of its
1H and 13C NMR spectroscopic data (Tables 1and 2) with
those of 1 and 2 implied that they were highly related in
structure, including their stereochemistry, with the excep-
C-2
C-3
C-4
168.5 168.2 168.5 168.8 167.9 171.4 168.0
112.8 112.8 112.9 112.7 112.6 114.1 112.7
178.4 178.2 178.4 179.4 178.4 182.0 178.5
C-5
C-6
C-7
C-8
64.0
66.6
74.5
70.0
64.0
66.6
74.5
70.0
64.0
66.7
74.6
70.1
68.8
73.2
73.5
70.0
64.7
72.7
70.6
68.4
66.7
74.0
72.5
70.1
64.7
72.7
70.7
68.4
1
tion of the phenylethyl part. The H NMR spectroscopic
C-9
162.2 162.2 162.2 161.6 163.1 165.3 163.1
121.4 121.4 121.4 120.1 120.7 121.8 120.7
132.5 139.9 131.8 132.5 132.6 134.1 130.1
115.6 128.2 129.3 115.6 115.6 116.5 129.2
146.3 128.3 113.8 146.3 146.3 147.7 115.1
146.1 126.1 157.7 146.0 146.0 147.6 155.6
112.2 128.3 113.8 112.2 112.2 113.0 115.1
118.7 128.2 129.3 118.7 118.7 120.6 129.2
C-10
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
C-6Ј
C-7Ј
C-8Ј
data showed the presence of an AAЈBBЈ coupling system
[δH = 7.15 (d, J = 8.4 Hz, 2 H, 2Ј,6Ј-H), 6.83 (d, J = 8.4 Hz,
2 H, 3Ј,5Ј-H) ppm] and a methoxy signal at δH = 3.70 ppm,
which corresponds to a 4Ј-methoxyphenyl group. This as-
signment was confirmed by analysis of the HMBC corre-
lations. Thus, compound
3
was determined as
31.2
34.3
31.7
34.0
–
31.0
34.4
55.0
31.1
34.3
55.6
31.1
34.3
55.6
33.1
36.4
56.5
31.2
34.5
–
(5S,6S,7S,8R)-2-[2-(4Ј-methoxyphenyl)ethyl]-5,6,7,8-tetra-
hydroxy-5,6,7,8-tetrahydrochromone.
OCH3 55.6
The molecular formula of aquilarone D (4) was deter-
mined as C18H20O8 from its HRMS (ESI) data, m/z =
365.1230 [M + H]+ (calcd. 365.1239). The 1H and 13C
NMR spectroscopic data of 4 (Tables 1 and 2) are highly
similar to those of 1, suggesting that they have the same
(3Ј-hydroxy-4Ј-methoxyphenyl)ethyl unit with only minor
differences in the 5,6,7,8-tetrahydrochromone part. De-
tailed comparison of their 13C NMR spectroscopic data re-
vealed that the only difference is the stereochemistry of 6-
OH. This conclusion was consistent with the different cou-
pling constants of J5,6 (7.0 Hz), J5,5-OH (3.0 Hz), J6,7
(7.0 Hz), J6,6-OH (4.5 Hz) in 4, and its comparatively large
coupling constants require the presence of 6α-H, instead
[a] Compounds 2 and 3 measured at 100 MHz, others at 125 MHz.
[b] Measured in CD3OD.
1
Figure 2. Key HMBC and H–1H COSY correlations of 1.
scopic data of 2 (Tables 1 and 2) are very similar to those of 6β-H as in 1. The deduction was supported further by
1
of 1, suggesting their similarity in structure, except for the comparing the H and 13C NMR spectroscopic data and
disappearance of the ABX coupling system and the meth- optical rotation value (–58.0 for 4, +18.0 for 1) with that of
oxy signal, and the appearance of symmetrical phenyl multi- isoagarotetrol (–58.6),[16,27] which confirms the 6α-H (6R)
plets at δH = 7.16–7.26 ppm (m, 5 H), consistent with differ- configuration for this molecule. Therefore, 4 was elucidated
ences in its 13C NMR spectroscopic data. Identical data for as (5S,6R,7S,8R)-2-[2-(3Ј-hydroxy-4Ј-methoxyphenyl)ethyl]-
the chromone moiety as well as the same sign of the optical 5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone.
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FULL PAPER
Figure 3. Synthetic route for derivatives 1a–1d from 1.
Figure 4. X-ray structure of 1a and CD spectrum of 1d.
Aquilarone E (5) was obtained as white needle-type crys- phenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochrom-
tals. Its molecular formula C18H20O8 was assigned from its one (Figure 1).
1
HRMS (ESI) data. Its IR, H NMR, and 13C NMR spec-
Aquilarone G (7) has the molecular formula C19H18O6
troscopic data (Tables 1 and 2) exhibited close similarities as determined from the HRMS (ESI) ion peak at m/z =
with those of 1, implying that they share the same 2-[2-(4Ј- 343.1176 [M + H]+ (calcd. 343.1181). The presence of hy-
hydroxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetra-
droxy (3415 cm–1) and α,β-unsaturated ketone (1636 cm–1)
1
hydrochromone planar structure, which was supported by moieties was evident from the IR spectrum. The H and
heteronuclear single quantum coherence (HSQC) and 13C NMR spectroscopic data (Table 3) showed signals for
HMBC spectra. Differences in the stereochemistry of 5-OH an ABX coupling system, two methylene groups, and a
and 6-OH were revealed after a careful comparison of their methoxy group, indicating the presence of a (3-methoxy-4-
1H and 13C NMR spectroscopic data, with different data of hydroxyphenyl)ethyl structure, which was supported by
δC = 72.7 (C-6), 70.6 (C-7), and 68.4 (C-8) ppm compared analysis of the HMBC correlations (Figure 5). Similarly,
to those of 1. A detailed analysis and comparison of the another set of signals of two aromatic singlets at δH = 7.23
1H and 13C NMR spectroscopic data, including coupling (5-H) and 7.11 (8-H) ppm, one olefinic proton at δH = 6.03
constants, with those of agarotetrol,[13,16] especially for the (3-H) ppm, another methoxy group, together with an α,β-
5,6,7,8-tetrahydrochromone part, suggested their identical unsaturated carbonyl group (C-4) implied the presence of a
stereochemistry. Thus, the structure of 5 was elucidated as chromone derivative unit with tetrasubstitution on the aro-
(5S,6R,7R,8S)-2-[2-(3Ј-hydroxy-4Ј-methoxyphenyl)ethyl]- matic ring [δH = 7.23 (s)/δC = 107.2(C-5), δH = 7.11 (s)/
5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone.
δC = 100.3(C-8), δC = 144.9 (C-6), δC = 153.4 (C-7) ppm].
Analysis of the HR mass (ESI) spectrum of aquilarone F Assignments of the chromone part, the substitution posi-
(6) gave its molecular formula as C17H18O7. The overall ap- tions of the methoxy and hydroxy groups, as well as the
pearance of its 1H and 13C NMR spectroscopic data complete structure of 7 were determined by detailed analy-
(Tables 1 and 2) suggests it is structurally related to 5, ex- sis of the HMBC correlations (Figure 5). Finally, 7 was elu-
cept for differences in the 2-(3Ј-hydroxy-4Ј-methoxyphenyl)- cidated as 4Ј,6-dihydroxy-3Ј,7-dimethoxy-2-(2-phenyl)ethyl-
ethyl part. Interpretation of its MS, 1H NMR, and 13C chromone.
NMR data, in combination with HMBC correlations, es-
The molecular formula of aquilarone H (8) was estab-
tablished its structure as (5S,6R,7R,8S)-2-[2-(4Ј-hydroxy- lished as C18H16O4 from the HRMS (ESI) ion peak at m/z
4
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Nine 2-(2-Phenylethyl)chromone Derivatives from Aquilaria sinensis
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Table 3. H and 13C NMR spectroscopic data (in [D6]DMSO) of compounds 7–9, δ in ppm (J in Hz).
No.
7[a]
δH
8[a]
δH
9[b]
δH
δC
δC
δC
2
3
4
5
6
7
8
9
10
1Ј
2Ј
3Ј
4Ј
5Ј
6Ј
7Ј
–
167.7
108.7
176.0
107.2
144.9
153.4
100.3
150.9
116.5
130.8
112.5
147.4
144.8
115.3
120.4
31.8
35.2
–
–
168.7
108.9
176.5
104.7
156.3
122.9
119.7
150.6
123.7
130.0
129.2
115.1
155.7
115.1
129.1
31.3
35.2
55.6
–
–
168.4
108.6
176.6
107.4
154.6
122.7
119.4
149.6
123.9
132.5
115.6
146.3
146.0
112.2
118.7
31.4
35.0
–
6.03 s
–
7.23 s
–
6.14 s
–
7.36 d (2.0)
–
7.34 dd (8.8, 2.0)
7.56 d (8.8)
–
–
6.10 s
–
7.24 d (2.0)
–
7.18 dd (8.8, 2.0)
7.47 d (8.8)
–
–
–
7.11 s
–
–
–
6.78 d (2.0)
–
–
–
–
7.01 d (8.5)
6.65 d (8.5)
–
6.65 d (8.5)
7.01 d (8.5)
2.88 m
2.87 m
3.82 s
–
–
–
6.64 d (2.0)
–
–
6.78 d (8.2)
6.58 dd (8.2, 2.0)
2.88 m
2.85 m
–
–
–
6.64 d (8.0)
6.60 dd (8.0, 2.0)
2.87 m
2.87 m
–
3.88 s
3.67 s
–
8Ј
6-OCH3
7-OCH3
3Ј-OCH3
4Ј-OCH3
3Ј-OH
4Ј-OH
6-OH
56.1
55.4
–
–
–
–
–
55.6
–
–
–
–
–
–
3.69 s
8.79 br. s
–
–
–
8.72 br. s
9.71 br. s
9.20 s
–
–
–
10.29 br. s
–
[a] Measured at 500/125 MHz. [b] Measured at 400/100 MHz.
HPLC Comparison Analysis
2-(2-Phenylethyl)chromone derivatives are considered
special, not only for their limited distribution in Aquilaria
(only 57 congeners were previously reported), but even
more for the fact that they uniquely exist in the resinous
wood and are believed to be produced either by decay or
injury of the stems. None of the 2-(2-phenylethyl)chromone
derivatives were discovered in the resin-free woods of Aquil-
aria species. An HPLC comparison of the resin-deposited
and resin-free woods provided further proof for this hy-
pothesis (Figure 6). Therefore, to some extent, the 2-(2-
phenylethyl)chromone derivatives in Aquilaria plants are
probably produced to fight against decay or disease. Vali-
dation of this hypothesis will be an interesting issue, al-
though the details of the resin deposition process in Aquila-
ria species is still not clearly understood.
Figure 5. Selected HMBC correlations of 7.
= 297.1121 [M + H]+ (calcd. 297.1130). The 1H NMR spec-
troscopic data (Table 3) showed the signals ascribed to an
ABX system [δH = 7.56 (d, 1 H, J = 8.8 Hz), 7.36 (d, 1 H,
J = 2.0 Hz), 7.34 (dd, 1 H, J = 8.8, 2.0 Hz) ppm] and an
AAЈBBЈ system [δH = 7.01 (d, 2 H, J = 8.5 Hz), 6.65 (d, 2
H, J = 8.5 Hz) ppm], one methoxy group at δH = 3.82 ppm,
and a broad signal due to one hydroxy group at δH
=
9.20 ppm, suggesting a 2-(2-phenylethyl)chromone deriva-
tive with one hydroxy and one methoxy substituent. HMBC
correlations from methoxy protons to C-6 determined its
attachment at C-6 (δ = 156.3 ppm). Consequently, 8 was
elucidated as 4Ј-hydroxy-6-methoxy-2-(2-phenylethyl)chro-
mone.
Biological Activities
Inspired by the report that the EtOH extract of A. si-
nensis leaves exhibited significant anti-inflammatory ac-
Aquilarone I (9) has the molecular formula C18H16O5 as tivity,[8] and the fact that NO plays an important role in
established from its HRMS (ESI) data. Interpretation of its the inflammatory process, inhibitors of NO release may be
1H and 13C NMR spectroscopic data (Table 3) allowed us considered as therapeutic agents for inflammatory dis-
to assign 9 also as a 2-(2-phenylethyl)chromone derivative, eases.[39,40] The inorganic free radical NO is synthesized by
substituted by a methoxy and two hydroxy groups. Analysis a family of enzymes termed NO-synthases (NOS), and its
of the HMBC correlations established the methoxy group cytotoxic properties are part of a host defense mechanisms
(δH = 3.69 ppm) at C-4Ј, and two hydroxy groups (δH
=
against pathogenic microorganisms.[39] However, excess pro-
8.79 and 10.29 ppm) at C-6 (δC = 154.6 ppm) and C-3Ј (δC duction of NO, due to the reaction with superoxide in bio-
= 146.3 ppm), respectively. Eventually, 9 was elucidated as logical systems, gives rise to various conditions such as in-
3Ј,6-dihydroxy-4Ј-methoxy-2-(2-phenylethyl)chromone.
flammation, carcinogenesis, and atherosclerosis.[40] There-
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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D. Chen, Z. Xu, X. Chai, K. Zeng, Y. Jia, D. Bi, Z. Ma, P. Tu
FULL PAPER
Figure 6. HPLC chromatograms of reference standards (A), ALR (B) and the stems of A. sinensis (C).
fore, inhibition of NO production is important to control 11) exhibited considerable activity at the same level. And
infection and NO-dependent inflammatory diseases. Based neither the different methoxy and hydroxy substitution pat-
on the above consideration, all compounds were tested for terns, nor their different substitution sites has a significant
their inhibitory effects against lipopolysaccharide (LPS) in- impact on the activity.
duced NO production in RAW 264.7 macrophages and
anti-COX-2 activity. The results showed that all assayed
compounds exhibit potent inhibitory activity against NO
production in RAW 264.7 cells, with IC50 values of 5.12–
22.26 μm; by comparison the positive control ibuprofen, a
clinical anti-inflammatory agent, has an IC50 value of
94.12 μm (Table 4). However, these compounds did not ex-
hibit obvious inhibition against COX-2.
Conclusions
Nine 2-(2-phenylethyl)chromone derivatives, namely,
aquilarones A–I (1–9), along with two known congeners (10
and 11) were isolated from the resin-deposited wood of
Aquilaria sinensis (Lour.) Gilg. The structures of the new
compounds were elucidated by means of NMR spec-
troscopy and MS techniques. The absolute configuration of
aquilarone A (1) was achieved by chemical methods and
Table 4. Inhibition against LPS-induced NO production in RAW
264.7 for 1–11.[a]
Compound
IC50 [μm]
IC50 [μm]
was confirmed by single-crystal X-ray diffraction analysis
of its acetonide derivative 1a. An anti-inflammatory assay
showed that these 2-(2-phenylethyl)chromone derivatives
exhibit significant inhibitory effects against NO production
in RAW 264.7 cells. Also included is a comparison analysis
by HPLC–UV for the resin-free and resinous woods of
Aquilaria sinensis, which – to some extent – provided impor-
tant information in understanding the biosynthesis of the
2-(2-phenylethyl)chromone derivatives in eaglewood.
1
2
3
4
5
6
9.03
5.12
7.71
7.49
22.26
13.09
7
7.94
5.95
7.59
7.94
6.59
94.12
8
9
10
11
Ibuprofen
[a] Data are presented as mean values based on three experiments.
Strcuture–Activity Relationship
Experimental Section
No obvious structure–activity relationship was found for
these compounds. Both 2-(2-phenyl)ethyltetrahydrochro-
General Experimental Procedures: Optical rotation measurements
mone derivatives (1–6) and 2-(2-phenyl)ethylchromones (7– were carried out with a Perkin–Elmer 243B digital polarimeter. UV
6
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Nine 2-(2-Phenylethyl)chromone Derivatives from Aquilaria sinensis
1
spectra were measured with a Shimadzu spectrometer and IR spec-
tra were measured with a Nicolet Avatar 360 FTIR spectrometer
as KBr pellets. CD spectra were recorded with a JACSO J-815 spec-
trometer. HRMS (ESI) were measured with an Auto Spec Ultima-
TOF mass spectrometer. NMR spectra were measured with a Var-
ian 500 apparatus at 500 MHz (1H) or 125 MHz (13C) and a Bruker
apparatus at 400 MHz (1H) or 100 MHz (13C) with tetramethylsil-
ane (TMS) as an internal standard. Preparative HPLC was per-
formed with a Waters 2487 instrument and an ODS column
(Allsphere, 250 mmϫ10 mm, i.d. 5 μm). Column chromatography
was performed with silica gel (100–200 and 200–300 mesh, Qingdao
Haiyang Chemical Co. Ltd., China), Sephadex LH-20 (40–70 μm;
Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Rp C18
gel (Merck, Darmstadt, Germany). The chemical ibuprofen (purity
Ն 98.0%) was purchased from Sigma.
nm. IR (KBr): ν = 3356, 1659, 1602, 1496, 1450 cm–1. H and 13C
˜
NMR: Tables 1 and 2. HRMS (ESI): calcd. for C17H19O6 [M +
H]+ 319.1174; found 319.1176.
Aquilarone C (3): Pale yellow amorphous powder. [α]2D0 = +12.0 (c
= 1.0, MeOH). UV (MeOH): λmax (logε) = 252 (4.17), 209 (4.27)
nm. IR (KBr): νmax = 3405, 3309, 1662, 1578, 1514, 1451 cm–1. 1H
˜
and 13C NMR: Tables 1 and 2. HRMS (ESI): calcd. for C18H21O7
[M + H]+ 349.1291; found 349.1281.
Aquilarone D (4): White needle-type crystals. M.p. 190–191 °C.
[α]2D0 = –58.0 (c = 1.0, MeOH). UV (MeOH): λmax (logε) = 253
(4.04), 213 (4.19) nm. IR (KBr): ν
= 3340, 1657, 1589,
˜
max
1515 cm–1. 1H and 13C NMR: Tables 1 and 2. HRMS (ESI): calcd.
for C18H21O8 [M + H]+ 365.1239; found 365.1230.
Aquilarone E (5): White needle-type crystals. M.p. 212–213 °C. [α]
20 = –16.0 (c = 1.0, MeOH). UV (MeOH): λmax (logε) = 281 (4.12),
D
Plant Material: Aquilariae lignum resinatum was collected in Sep-
tember 2009 in Hainan Province, China. The identification of the
material was performed by Professor Peng-Fei Tu. A voucher speci-
men (KDC 20090901) is deposited at the Herbarium of the Modern
Research Center for Traditional Chinese Medicine, Peking Univer-
sity Health Science Center.
252 (4.23), 212 (4.04) nm. IR (KBr): ν
= 3357, 1656.9, 1590,
˜
max
1514, 1447 cm–1. H and 13C NMR: Tables 1 and 2. HRMS (ESI):
calcd. for C18H21O8 [M + H]+ 365.1233; found 365.1230.
1
Aquilarone F (6): Pale yellow amorphous powder. [α]2D0 = –15.0 (c
= 1.0, MeOH). UV (MeOH): λmax (logε) = 252 (4.07), 213 (4.23)
1
nm. IR (KBr): ν
= 3381, 1657, 1597, 1515, 1445 cm–1. H and
˜
max
Extraction and Isolation: The ALR material (3 kg) was exhaustively
extracted with CHCl3 (2ϫ10 L) and 95% EtOH (2ϫ10 L) by suc-
cessive soxhlet extractions. The CHCl3 extract (140 g) was sub-
jected to a silica gel chromatography column (CC) (10ϫ120 cm,
100–200 mesh) and eluted with a gradient of petroleum ether (PE)/
EtOAc (50:1–1:1, v/v) to yield eight fractions (C1–C8). Fraction C5
(10 g) was applied to a silica gel CC (8ϫ70 cm, 200–300 mesh) and
eluted with PE/acetone (4:1) to give fractions C5-1–C5-8. Frac-
tion C5-6 (1.2 g) was then separated by using semipreparative
HPLC (MeOH/H2O, 75:25, 2 mL/min) to yield 7 (4.4 mg, tR
22.0 min). Fraction C6 (5 g) was subjected to a silica gel CC
(5ϫ50 cm, 200–300 mesh) and eluted with PE/acetone (4:1–1:1,
4 L) to give eight fractions (C6-1–C6-8). Fraction C6-4 (1.2 g) was
purified by semipreparative HPLC (MeOH/H2O, 60:40, 2 mL/min)
to give 8 (225 mg, tR = 34.2 min). Fraction C6-5 (0.8 g) was puri-
fied by using an octadecylsilane (ODS) CC (2ϫ50 cm, 20 g) and
eluted with MeOH/H2O (60:40) to give 9 (16 mg). The 95% EtOH
extract (150 g) was subjected to a silica gel CC (10ϫ120 cm, 100–
200 mesh) and eluted with a gradient of CHCl3/MeOH (30:1–2:1,
v/v) to yield six fractions (E1–E6). Fraction E4 (11 g) was subjected
to an ODS CC (4ϫ40 cm) and eluted with a gradient of MeOH/
H2O (1:4–1:1, 3.0 L) to give 58 fractions (E4-1–E4-58). A portion
of combined fractions E4-17–E4-32 (2.4 g) was purified by semipre-
parative HPLC (ACN/H2O, 12:88, 2 mL/min) to give 2 (17 mg, tR
24 min) and 6 (5 mg, tR 32 min). Fraction E5 (10 g) was subjected
to a silica gel CC (8ϫ 70 cm, 100–200 mesh) and eluted with a
gradient of CHCl3/MeOH (10:1–6:1, 8.0 L) to give 62 fractions
(E5-1–E5-62). Fractions E5-22–E5-48 were combined (3.2 g) and
purified by using semipreparative HPLC (MeOH/H2O, 25:75,
2 mL/min) to give 2 (60 mg, tR = 32 min), 4 (70 mg, tR = 23 min),
and 1 (60 mg, tR = 40 min). Fractions E5-49–E5-57 were combined
(0.5 g) and purified by using semipreparative HPLC (MeOH/H2O,
25:75, 2 mL/min) to yield 5 (36 mg, tR = 60 min). The purities of
these compounds are above 98% as determined by HPLC.
13C NMR: Tables 1 and 2. HRMS (ESI): calcd. for C17H19O7 [M
+ H]+ 335.1125; found 335.1124.
Aquilarone G (7): Pale yellow amorphous powder. UV (MeOH):
λmax (logε) = 210 (4.54), 231 (4.43), 281 (4.02), 322 (3.99) nm. IR
(KBr): ν
= 3415, 1636, 1567, 1518, 1459 cm–1. 1H and 13C
˜
max
NMR: Table 3. HRMS (ESI): calcd. for C19H19O6 [M + H]+
343.1181; found 343.1176.
Aquilarone H (8): White amorphous powder. UV (MeOH): λmax
(logε) = 226 (4.43), 240 (4.41), 328 (3.74) nm. IR (KBr): ν
=
˜
max
3339, 1633, 1606, 1592, 1482 cm–1. 1H and 13C NMR: Table 3.
HRMS (ESI): calcd. for C18H17O4 [M + H]+ 297.1130; found
297.1121.
Aquilarone I (9): Yellow amorphous powder. UV (MeOH): λmax
(logε) = 226 (4.46), 274 (3.92), 328 (3.78) nm. IR (KBr): ν
=
˜
max
3061, 1628, 1584, 1505, 1475 cm–1. 1H and 13C NMR: Table 3.
HRMS (ESI): calcd. for C18H17O5 [M + H]+ 313.1075; found
313.1070.
Acetonide Derivative of 1 (1a): To a suspension of 1 (9.4 mg,
0.026 mmol) in dry acetone (2.1 mL) and 2,2-dimethoxypropane
(0.7 mL) was added
a catalytic amount of PPTS (1.0 mg,
0.004 mmol), and the resultant reaction mixture was stirred at
room temperature until the starting material was consumed (moni-
tored by TLC). The solvent was removed under reduced pressure.
Purification by using preparative TLC afforded 1a as a colorless oil
(10.4 mg), which was recrystallized from MeOH to give colorless
prismatic crystals. [α]2D0 = +18.0 (c = 1.0, MeOH). 1H NMR
(400 MHz, CD3OD): δH = 6.03 (s, 1 H, 3-H), 5.14 (d, J = 5.5 Hz,
1 H, 5-H), 4.55 (dd, J = 5.5, 2.5 Hz, 1 H, 6-H), 3.72 (dd, J = 8.5,
2.5 Hz, 1 H, 7-H), 4.60 (d, J = 8.5 Hz, 1 H, 8-H), 6.61 (d, J =
2.0 Hz, 1 H, 2Ј-H), 6.76 (d, J = 8.4 Hz, 1 H, 5Ј-H), 6.55 (dd, J =
8.4, 2.0 Hz, 1 H, 6Ј-H), 2.82 (m, 4 H, 7Ј,8Ј-H), 1.32 (s, 3 H, CH3),
1.17 (s, 3 H, CH3), 3.73 (s, 3 H, OCH3) ppm. 13C NMR (100 MHz,
CD3OD): δC = 171.2 (C-2), 114.4 (C-3), 181.0 (C-4), 71.1 (C-5),
76.6 (C-6), 72.9 (C-7), 68.3 (C-8), 164.3 (C-9), 120.6 (C-10), 134.0
(C-1Ј), 116.4 (C-2Ј), 147.6 (C-3Ј), 147.6 (C-4Ј), 120.5 (C-5Ј), 112.8
(C-6Ј), 33.0 (C-7Ј), 36.3 (C-8Ј), 56.4 (4Ј-OCH3), 110.8, 26.1, 27.7
[C(CH3)2] ppm. HRMS (ESI): calcd. for C21H25O8 [M + H]+
405.1541; found 405.1544.
Aquilarone A (1): White amorphous powder. [α]2D0 = +18.0 (c = 1.0,
MeOH). UV (MeOH): λmax (logε) = 253 (3.87), 207 (4.22) nm. IR
(KBr): ν
= 3359, 1656, 1589, 1515, 1446 cm–1. 1H and 13C
˜
max
NMR: Tables 1 and 2. HRMS (ESI): calcd. for C18H21O8 [M +
H]+ 365.1240; found 365.1230.
Aquilarone B (2): Pale yellow amorphous powder. [α]2D0 = +13.1 (c
Compound 1b: A solution of 1a (10.1 mg) and 4-(dimethylamino)-
= 1.0, MeOH). UV (MeOH): λmax (logε) = 252 (4.07), 209 (4.24) pyridine (0.9 mg) in pyridine (1.0 mL) and acetic anhydride
Eur. J. Org. Chem. 0000, 0–0
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D. Chen, Z. Xu, X. Chai, K. Zeng, Y. Jia, D. Bi, Z. Ma, P. Tu
(1.0 mL) was stirred at room temperature for 5 h. The solvent was by using full-matrix least-squares difference Fourier techniques. All
FULL PAPER
removed under reduced pressure. The residue was purified by pre-
parative TLC (CH2Cl2/MeOH, 15:1) to give 1b (9.6 mg) as a color-
less oil. 1H NMR (400 MHz, CDCl3): δH = 6.11 (s, 1 H, 3-H), 5.30
(d, J = 5.5 Hz, 1 H, 5-H), 4.67 (dd, J = 5.5, 2.0 Hz, 1 H, 6-H), 5.32
non-hydrogen atoms were refined anisotropically, placed in ideal-
ized positions, and refined as riding atoms with relative isotropic
parameters. Crystal data of 1a: Monoclinic, C21H24O8, space group
P21, a = 8.120(3) Å, b = 19.967(4) Å, c = 12.413(3) Å, β =
(dd, J = 6.0, 2.0 Hz, 1 H, 7-H), 6.18 (dd, J = 6.0, 2.0 Hz, 1 H, 8- 91.14(1)°, V = 2012.3(1) Å3, Z = 4, Dcalcd. = 1.335 g/cm3, crystal
H), 6.82 (d, J = 2.4 Hz, 1 H, 2Ј-H), 6.87 (d, J = 8.4 Hz, 1 H, 5Ј-
H), 6.94 (dd, J = 8.4, 2.0 Hz, 1 H, 6Ј-H), 2.75–2.85 (m, 4 H, 7Ј-H,
8Ј-H), 1.34, 1.25 [s, 6 H, C(CH3)2], 2.18 (s, 6 H, COCH3), 2.30 (s,
3 H, COCH3), 3.80 (s, 3 H, OCH3) ppm. 13C NMR (100 MHz,
CDCl3): δC = 170.3 (C-2), 114.2 (C-3), 177.5 (C-4), 70.3 (C-5), 72.7
size 0.12ϫ0.14ϫ0.36 mm, 7028 independent reflections. The final
indices were R1 = 0.0519, wR2 = 0.1303, S = 1.014. CCDC-867726
contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_requ-
(C-6), 69.8 (C-7), 65.6 (C-8), 156.4 (C-9), 120.5 (C-10), 131.6 (C- est/cif.
1Ј), 122.6 (C-2Ј), 139.7 (C-3Ј),149.8 (C-4Ј), 126.3 (C-5Ј), 112.6 (C-
HPLC Procedure: The analyses were performed with an Agilent
6Ј), 31.5 (C-7Ј), 35.2 (C-8Ј), 56.4 (OCH3), 110.8, 25.9, 27.5
[C(CH3)2], 169.7, 20.8 (COCH3), 168.9, 20.6 (COCH3), 168.7, 20.5
(COCH3) ppm. HRMS (ESI): calcd. for C27H31O11[M + H]+
531.1867; found 531.1860.
Eclipse XDB C18 Column (250ϫ4.6 mm, i.d. 5 μm) at a column
temperature of 30 °C. The mobile phase consisted of 0.1% phos-
phoric acid in water (A) and acetonitrile (B) by using a gradient
program of 10–20% B in 0–20 min, 20–25% B in 20–35 min, 25–
35% B in 35–50 min, 35% B in 50–65 min. The flow rate was
1.0 mL/min, and the diode array detector (DAD) was set at 240 nm
for acquiring chromatograms.
Compound 1c: To a solution of 1b (6.8 mg) in dichloromethane
(1.0 mL), TFA (0.2 mL) was added dropwise at 0 °C. The reaction
mixture was then stirred at room temperature until the starting
material was consumed (monitored by TLC). The solvent was re-
moved under reduced pressure, and the residue was purified by
preparative TLC (CH2Cl2/MeOH, 15:1) to give 1c (5.8 mg) as a
colorless oil. 1H NMR (400 MHz, CDCl3): δH = 6.09 (s, 1 H, 3-
H), 4.96 (dd, J = 3.6, 2.0 Hz, 1 H, 5-H), 4.35 (dd, J = 3.6, 2.0 Hz,
1 H, 6-H), 5.23 (dd, J = 8.8, 2.0 Hz, 1 H, 7-H), 6.22 (dd, J = 8.8,
2.0 Hz, 1 H, 8-H), 6.81 (d, J = 2.0 Hz, 1 H, 2Ј-H), 6.87 (d, J =
8.4 Hz, 1 H, 5Ј-H), 6.94 (dd, J = 8.4, 2.0 Hz, 1 H, 6Ј-H), 2.85 (m,
4 H, 7Ј-H, 8Ј-H), 2.12 (s, 6 H, COCH3), 2.29 (s, 3 H, COCH3),
3.79 (s, 3 H, OCH3) ppm. 13C NMR (100 MHz, CDCl3): δC = 170.2
(C-2), 113.5 (C-3), 181.0 (C-4), 67.5 (C-5), 69.2 (C-6), 71.3 (C-7),
66.2 (C-8), 157.2 (C-9), 119.2 (C-10), 131.5 (C-1Ј), 122.6 (C-2Ј),
139.8 (C-3Ј),149.9 (C-4Ј), 126.2 (C-5Ј), 112.6 (C-6Ј), 31.5 (C-7Ј),
35.2 (C-8Ј), 55.9 (OCH3), 169.7, 20.8 (COCH3), 168.9, 20.6
(COCH3), 168.7, 20.5 (COCH3) ppm. HRMS (ESI): calcd. for
C24H27O11 [M + H]+ 491.1558; found 491.1547.
Preparation of Sample Solutions: The dried material was pulverized
to 80 mesh. The powder (ca. 1.0 g) was suspended in methanol
(10 mL) in a conical flask, accurately weighed, then sonicated for
1 h; the resultant suspension was cooled to room temperature. A
certain amount of methanol was added to the original weight ex-
tract. The final solution was filtered through a 0.22 μm filter, and
the primary filtrate was discarded. The subsequent filtrate was
stored as the sample solution until analysis.
Preparation of Standard Solutions and the Identification of Compo-
nents: These reference compounds were accurately weighed and dis-
solved in methanol and then diluted to appropriate concentration.
The mixed stock solution of standards, containing compound 1
(0.226 mgmL–1),
(0.230 mgmL–1),
(0.264 mgmL–1),
2
5
8
(0.265 mgmL–1),
(0.236 mgmL–1),
(0.285 mgmL–1),
3
6
9
(0.262 mgmL–1),
(0.342 mgmL–1),
4
7
(0.274 mgmL–1), 10
(0.264 mgmL–1), and 11 (0.265 mgmL–1), was prepared with meth-
anol. Peaks were identified by comparison of retention time and
DAD spectra with those of the corresponding standards. All stock
and working standard solutions were stored at 4 °C until used for
analysis.
Compound 1d: p-Methoxybenzoyl chloride (50.0 mg) was slowly
added to a pyridine solution (1.0 mL) of 1c at 0 °C, and the re-
sulting reaction mixture was stirred at room temperature overnight.
The solvent was removed under reduced pressure, and the residue
was purified by preparative TLC (CH2Cl2/MeOH, 15:1) to give 1d
(3.1 mg) as a colorless oil. [α]2D0 = +120.0 (c = 1.0, MeOH). 1H
NMR (400 MHz, CD3OD): δH = 6.12 (s, 1 H, 3-H), 7.82 (d, J =
8.4 Hz, 4 H), 7.79 (d, J = 8.4 Hz, 4 H), 6.00 (d, J = 5.5 Hz, 1 H,
5-H), 5.56 (dd, J = 5.5, 2.4 Hz, 1 H, 6-H), 5.70 (dd, J = 4.8, 2.4 Hz,
1 H, 7-H), 6.10 (d, J = 4.8 Hz, 1 H, 8-H), 6.87 (d, J = 2.0 Hz, 1
H, 2Ј-H), 6.94 (d, J = 8.4 Hz, 1 H, 5Ј-H), 6.94 (dd, J = 8.4, 2.0 Hz,
1 H, 6Ј-H), 2.88 (m, 4 H, 7Ј-H, 8Ј-H), 2.01 (s, 3 H, COCH3), 2.09
(s, 3 H, COCH3), 2.15 (s, 3 H, COCH3), 3.72 (s, 3 H, OCH3), 3.76
(s, 3 H, OCH3), 3.77 (s, 3 H, OCH3) ppm. 13C NMR (100 MHz,
CD3OD): δC = 171.2 (C-2), 113.8 (C-3), 179.1 (C-4), 68.4 (C-5),
68.9 (C-6), 70.2 (C-7), 63.0 (C-8), 160.3 (C-9), 122.6 (C-10), 132.8
(C-1Ј), 122.6 (C-2Ј), 141.1 (C-3Ј), 151.2 (C-4Ј), 127.6 (C-5Ј), 114.7
(C-6Ј), 32.5 (C-7Ј), 35.8 (C-8Ј), 56.4 (OCH3), 56.0 (OCH3) 171.3,
20.8 (COCH3), 170.9, 20.5 (COCH3), 170.2, 20.5 (COCH3), 166.5,
165.4, 133.4, 114.8, 123.1, 133.4, 114.8 (Ph), 166.3, 165.3, 132.8,
114.7, 123.1, 132.8, 114.7 (Ph) ppm. HRMS (ESI): calcd. for
C40H39O15 [M + H]+ 759.2285; found 759.2283.
Assay for Inhibition against LPS-Induced NO Production: RAW
264.7 cells grown on a 100 mm culture dish were harvested and
seeded in 48-well plates at 6ϫ104 cells/well for NO assay. The cells
were pretreated with various concentrations of samples for 30 min
and then incubated for 24 h with or without 1 μg/mL of LPS. The
nitrite concentration in the culture supernatant was measured by
the Griess reaction. Cell viability was measured by an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] as-
say (Sigma–Aldrich).[36]
Supporting Information (see footnote on the first page of this arti-
cle): 1D, 2D NMR and HR mass (ESI) spectra for 1–9 and 1a–1d.
Acknowledgments
The work was financially supported by projects of the Formulation
of Pharmacopeia of China (Edition 2015), the Special Program for
New Drug Innovation of the Ministry of Science and Technology,
China (2009ZX311-004, 2009ZX0308-004), and the National Natu-
ral Science Foundation of China (Grant No. 30873072). We are
also grateful to scientists of the Analytical Center of the Peking
University Health Science Center.
X-ray Crystallographic Analysis of 1a: Crystal data were obtained
with a Rigaku MicroMax 002+ CCD single-crystal diffractometer
with Cu-Kα radiation operating in the ω- and κ-scan mode. The
structure was solved by direct methods (SHELXS-97) and refined
8
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Nine 2-(2-Phenylethyl)chromone Derivatives from Aquilaria sinensis
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Received: May 30, 2012
Published Online: ᭿
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9

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Date: 13-08-12 12:22:23
Pages: 10
D. Chen, Z. Xu, X. Chai, K. Zeng, Y. Jia, D. Bi, Z. Ma, P. Tu
FULL PAPER
Natural Products
D. Chen, Z. Xu, X. Chai, K. Zeng, Y. Jia,
D. Bi, Z. Ma, P. Tu* ...................... 1–10
Nine 2-(2-Phenylethyl)chromone Deriva-
tives from the Resinous Wood of Aquilaria
sinensis and Their Inhibition of LPS-In-
duced NO Production in RAW 264.7 Cells
Keywords: Biological activity / Chromones /
Configuration determination
/ Anti-in-
flammatory agents / Eaglewood / Natural
products
Nine new 2-(2-phenylethyl)chromone de-
rivatives were isolated from the resin-de-
posited wood of Aquilaria sinensis. Their
structures were elucidated by means of
NMR spectroscopy, mass spectrometry, X-
ray diffraction, and chemical methods.
These compounds exhibit significant inhi-
bition of LPS-induced NO production in
RAW 264.7 cells.
10
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