Anti-inflammatory benzenoids from Antrodia camphorata

Article abstract of DOI:10.1021/np070045e

Four new compounds, including three new benzenoids, antrocamphin A (1), antrocamphin B (2), and 2,3,4,5-tetramethoxybenzoyl chloride (3), and a new 1,3-dioxolan-2-one derivative, antrodioxolanone (4), together with 13 known compounds have been isolated fr

Full text of DOI:10.1021/np070045e

J. Nat. Prod. 2007, 70, 989-992
Anti-inflammatory Benzenoids from Antrodia camphorata
Jih-Jung Chen,*,† Wen-Jiou Lin,† Chang-Hui Liao,‡ and Po-Chuen Shieh†
989
Graduate Institute of Pharmaceutical Technology, Tajen UniVersity, Pingtung 907, Taiwan, Republic of China, and Graduate Institute of
Natural Products, Chang Gung UniVersity, Taoyuan 333, Taiwan, Republic of China
ReceiVed January 31, 2007
Four new compounds, including three new benzenoids, antrocamphin A (1), antrocamphin B (2), and 2,3,4,5-
tetramethoxybenzoyl chloride (3), and a new 1,3-dioxolan-2-one derivative, antrodioxolanone (4), together with 13
known compounds have been isolated from the fruiting body of Antrodia camphorata. The structures of these new
compounds were determined through spectral analyses including extensive 2D-NMR data. Among the isolates,
antrocamphin A (1), antcin A (10), and antcin B (11) exhibited potent inhibition against fMLP-induced superoxide
production with IC50 values less than 10 µM.
Antrodia camphorata Wu, Ryvarden & Chang (Polyporaceae,
Aphyllophorales)1 is a parasitic fungus on the inner heartwood wall
of the endemic species Cinnamomum kanehirai Hay (Lauraceae)
in Taiwan. The fruiting body of A. camphorata is well known in
Taiwan by the name niu-chang-chih or jang-jy and is also popular
and very expensive as a medicinal material. It has been used for
the treatment of food, alcohol, and drug intoxication, diarrhea,
abdominal pain, hypertension, itching, and liver cancer in Chinese
folk medicine.2 Previous chemical studies of the fruiting body of
this fungus have reported the isolation of several components
including phenyl derivatives, triterpenoids, diterpenes, sesquiter-
penoids, steroids, lignans, fatty acids, and maleic and succinic acid
derivatives.3-13 Cytotoxic,11 anti-inflammatory,12 and neuroprotec-
tive13 activities have been demonstrated for some of these com-
pounds. In a screening program searching for anti-inflammatory
compounds in Formosan fungi, A. camphorata was shown to be
one of the active species. Investigation on the n-hexane-soluble
fraction of the fruiting body of A. camphorata has led to the
isolation and characterization of three new benzenoids, antrocam-
phin A (1), antrocamphin B (2), and 2,3,4,5-tetramethoxybenzoyl
chloride (3), and a new 1,3-dioxolan-2-one derivative, antrodiox-
Figure 1. NOESY (a) and HMBC (b) correlations of 1.
olanone (4), and 13 known compounds. This paper describes the
structural elucidation of 1-4 and the anti-inflammatory activities
of the isolates.
C-2 and between Me-3 and C-2 and C-4. Thus, the structure of 1
was elucidated as 1,2,5-trimethoxy-3-methyl-4-(3-methylbut-3-en-
1-ynyl)benzene, named antrocamphin A. This was further confirmed
by 1H-1H COSY and NOESY experiments (Figure 1). The
assignment of 13C NMR resonances was confirmed by HSQC and
HMBC techniques (Figure 1).
Extensive chromatographic purification of the n-hexane-soluble
fraction of the fruiting body of A. camphorata on a silica gel column
and preparative TLC afforded four new (1-4) and 13 known
compounds (5-17).
Results and Discussion
Antrocamphin A (1) was isolated as a yellowish oil. The
HRESIMS gave an [M + H]+ ion at m/z 247.1337, consistent with
the molecular formula C15H19O3. The IR spectrum showed a CtC
stretch at 2196 cm-1 and an aromatic ring CdC stretch at 1593,
1
1490, and 1462 cm-1. The H NMR spectrum of 1 showed the
presence of three methoxy groups, an aromatic methyl group, an
aromatic proton, and a 3-methylbut-3-en-1-ynyl group. On the basis
of the NOESY correlations (Figure 1) between H-6 and OMe-1
and OMe-5 and between OMe-5 and Me-3′ of 3-methylbut-3-en-
1-ynyl group, the 3-methylbut-3-en-1-ynyl group was assigned to
reside at C-4. The assignments of OMe-2 and Me-3 were confirmed
by HMBC correlations (Figure 1) between H-6 and OMe-2 and
* To whom correspondence should be addressed. Tel: +886-8-7624002,
ext. 332. Fax: +886-8-7625308. E-mail: jjchen@mail.tajen.edu.tw.
† Tajen University.
‡ Chang Gung University.
10.1021/np070045e CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/09/2007

990 Journal of Natural Products, 2007, Vol. 70, No. 6
Chen et al.
Figure 3. NOESY (a) and HMBC (b) correlations of 3.
Figure 2. NOESY (a) and HMBC (b) correlations of 2.
Antrocamphin B (2) was isolated as a yellowish powder. The
EIMS afforded the molecular ion [M]+ at m/z 248, implying a
molecular formula of C14H16O4, which was confirmed by the
HRESIMS. The IR spectrum showed a CtC stretch at 2182 cm-1
1
and a carbonyl absorption at 1661 cm-1. The H NMR spectrum
of 2 was similar to that of antrocamphin A (1), except that a
3-oxobut-1-ynyl group of 2 replaced the 3-methylbut-3-en-1-ynyl
group of 1. Analysis of the 1H NMR spectrum of 2 showed
resonances for an aromatic proton, three aromatic methoxy groups,
an aromatic methyl group, and a 3-oxobut-1-ynyl group. In the
NOESY spectrum of 2, the proton at δ 6.32 (H-6) showed
correlations with the methoxy resonances at δ 3.89 (OMe-1) and
3.92 (OMe-5), and the methoxy at δ 3.92 (OMe-5) with Me-3′ (δ
2.46) of the 3-oxobut-1-ynyl group. Thus, the 3-oxobut-1-ynyl group
was assigned to reside at C-4. Furthermore, the assignments of
OMe-2 and Me-3 were confirmed by HMBC correlations (Figure
2) between H-6, OMe-2, and C-2 and between Me-3 and C-2 and
C-4. On the basis of the above data, the structure of 2 was elucidated
as 4-(3,4,6-trimethoxy-2-methylphenyl)but-3-yn-2-one, named antro-
camphin B. This was further confirmed by 1H-1H COSY and
NOESY (Figure 2) experiments. The assignment of 13C NMR
resonances was confirmed by DEPT, HSQC, and HMBC (Figure
2) techniques.
Figure 4. NOESY (a) and HMBC (b) correlations of 4.
methanolysis experiment of 3 to give methyl 2,3,4,5-tetramethoxy-
1
benzoate (3a), which was confirmed by H NMR and HREIMS
data.
2,3,4,5-Tetramethoxybenzoyl chloride (3) was isolated as an
amorphous powder with a molecular formula of C11H13ClO5 as
determined by positive-ion HRESIMS and 13C NMR data. The
presence of a carbonyl group was revealed by a band at 1768 cm-1
in the IR spectrum, which was confirmed by the resonances at δ
168.1 in the 13C NMR spectrum. Comparison of the IR and 1H and
13C NMR data of 3 with those of 2,3,4,5-tetramethoxybenzoic acid14
suggested that their structures are closely related except that the
chlorocarbonyl group of 3 replaced the carboxy group of 2,3,4,5-
tetramethoxybenzoic acid. Analysis of the 1H NMR spectrum of 3
showed resonances for an aromatic proton and four aromatic
methoxy groups. A lower field aromatic proton at δ 7.13 suggested
that it was adjacent to the chlorocarbonyl group. In the NOESY
spectrum of 3, the proton at δ 7.13 (H-6) showed correlations with
the methoxy resonance at δ 3.91 (OMe-5). Thus, the other three
methoxy groups were assigned to C-2, C-3, and C-4. On the basis
of the above data, the structure of 3 was elucidated as 2,3,4,5-
tetramethoxybenzoyl chloride. This was further confirmed by 1H-
1H COSY and NOESY (Figure 4) experiments. The assignment of
13C NMR resonances was confirmed by DEPT, HSQC, and HMBC
(Figure 4) techniques. This is the first report of the occurrence of
3 in a natural source, although it has been used as a reagent.15
Further evidence to support the structure of 3 was also sought in a
Antrodioxolanone (4) was obtained as a yellowish, amorphous
solid, and the molecular formula was confirmed to be C29H32O9
from the pseudomolecular ion peak at m/z ) 547.1946 [M + Na]+
obtained by HRESIMS. In the IR spectrum, absorptions for alkyne
(2186 cm-1), 1,3-dioxolan-2-one carbonyl (1765 cm-1), and
aromatic olefinic functions (1598, 1497, and 1455 cm-1) were
observed. In the 1H and 13C NMR spectra, the number of resonances
observed was half that expected, suggesting that 4 had a sym-
metrical structure. The 1H NMR spectrum of 4 was similar to that
of 2 except that the C-2′ acetyl group of 2 was replaced by a 4,5-
dimethyl-2-oxo-5-((3,4,6-trimethoxy-2-methylphenyl)ethynyl)-1,3-
dioxolan-4-yl group of 4. The formation of 4 may be the result of
intermolecular cyclization16 at the acetyl group of 2 to give a cyclic

Anti-inflammatory Benzenoids from Antrodia camphorata
Journal of Natural Products, 2007, Vol. 70, No. 6 991
Experimental Section
Table 1. IC50 Values of Compounds Isolated from the Fruiting
Body of A. camphorata in the Inhibition on fMLP-Induced
Superoxide Generation in Human Neutrophils
General Experimental Procedures. All melting points were
determined on a Yanaco micro-melting point apparatus and were
uncorrected. IR spectra (KBr or neat) were recorded on a Perkin-Elmer
system 2000 FT-IR spectrometer. Optical rotations were measured using
a Jasco DIP-370 polarimeter in CHCl3. UV spectra were obtained on
a Jasco UV-240 spectrophotometer. EI, ESI, and HRESI mass spectra
were recorded on a Bruker APEX II mass spectrometer. HREI, FAB,
and HRFAB mass spectra were recorded on a JEOL JMX-HX 110
mass spectrometer. NMR spectra, including COSY, NOESY, HMBC,
and HSQC experiments, were recorded on a Varian Unity 400 or a
Varian Inova 500 spectrometer operating at 400 and 500 MHz (1H)
and 100 and 125 MHz (13C), respectively, with chemical shifts given
in ppm (δ) using TMS as an internal standard. Silica gel (70-230,
230-400 mesh) (Merck) was used for CC. Silica gel 60 F-254 (Merck)
was used for TLC and preparative TLC.
compound
IC50 (µM)a
antrocamphin A (1)
9.33 ( 3.31
antrocamphin B (2)
2,3,4,5-tetramethoxybenzoyl chloride (3)
antrodioxolanone (4)
>100
>100
>100
4,7-dimethoxy-5-methyl-1,3-benzodioxole (5)
2,2′,5,5′-tetramethoxy-3,4,3′,4′-bimethylene-
dioxy-6,6′-dimethylbiphenyl (6)
(-)-sesamin (7)
26.09 ( 3.34
>100
90.23 ( 8.11
R-tocospiro B (8)
>100
>100
epi-friedelinol (9)
antcin A (10)
8.55 ( 1.04
antcin B (11)
9.82 ( 4.40
dehydroeburicoic acid (12)
ergosta-4,6,8(14),22-tetraen-3-one (13)
eburicol (14)
77.50 ( 7.43
>100
Plant Material. The fruiting body of A. camphorata was collected
from Nantou County, Taiwan, in December 2004. A voucher specimen
(Chen 6001) was deposited in the herbarium of the Department of
Pharmacy, Tajen University, Pingtung, Taiwan.
50.47 ( 2.84
>100
>100
â-sitostenone (15)
mixture of â-sitosterol (16) and
stigmasterol (17)
Extraction and Separation. The air-dried fruiting bodies of A.
camphorata (200 g) were extracted with n-hexane (3 × 2 L, each 24
h), EtOAc (3 × 2 L, each 24 h), and MeOH (3 × 2 L, each 24 h)
successively, and the extracts concentrated under reduced pressure. The
n-hexane extract (fraction A, 15 g) was chromatographed on silica gel
(70-230 mesh, 1.8 kg), eluting with n-hexane, gradually increasing
the polarity with EtOAc and MeOH to give 17 fractions: A1 (2.5 L,
n-hexane), A2 (1.5 L, n-hexane-EtOAc, 100:1), A3/A4 (each 2 L,
n-hexane-EtOAc, 80:1), A5 (1.5 L, n-hexane-EtOAc, 60:1), A6/A7
(each 1.5 L, n-hexane-EtOAc, 40:1), A8/A9 (each 2 L, n-hexane-
EtOAc, 30:1), A10/A11 (each 2.5 L, n-hexane-EtOAc, 20:1), A12
(1.5 L, n-hexane-EtOAc, 10:1), A13 (2.5 L, n-hexane-EtOAc, 5:1),
A14 (2 L, n-hexane-EtOAc, 3:1), A15 (2.5 L, n-hexane-EtOAc, 1:1),
A16 (3 L, EtOAc), A17 (4 L, MeOH). Fraction A2 (0.6 g) was
chromatographed further on silica gel (230-400 mesh, 90 g) eluting
with n-hexane-EtOAc (20:1) to give five fractions (each 1 L, A2-1-
A2-5). Fraction A2-3 (155 mg) was purified further by preparative TLC
(n-hexane-acetone, 30:1) to obtain 5 (13.8 mg) (Rf ) 0.35). Fraction
A5 (0.9 g) was chromatographed further on silica gel (230-400 mesh,
100 g) eluting with n-hexane-acetone (10:1) to give six fractions (each
1 L, A5-1-A5-6). Fraction A5-2 (115 mg) was purified further by
preparative TLC (n-hexane-acetone, 3:1) to obtain 15 (6.8 mg) (Rf )
0.67). Fraction A5-3 (196 mg) was purified further by preparative TLC
(n-hexane-acetone, 10:1) to obtain 16 and 17 (15.5 mg) (Rf ) 0.40).
Fraction A5-5 (127 mg) was purified further by preparative TLC (n-
hexane-acetone, 3:1) to obtain 7 (2.1 mg) (Rf ) 0.27). Fractions A6
and A7 (1.5 g) were chromatographed further on silica gel (230-400
mesh, 110 g) eluting with n-hexane-acetone (5:1) to give seven
fractions (each 1.5 L, A6-1-A6-7). Fraction A6-2 (75 mg) was purified
further by preparative TLC (CHCl3-EtOAc, 60:1) to obtain 8 (2.8 mg)
(Rf ) 0.68) and 14 (8.5 mg) (Rf ) 0.38). Fraction A6-3 (50 mg) was
purified further by preparative TLC (n-hexane-acetone, 3:1) to obtain
1 (6.8 mg) (Rf ) 0.58). Fraction A6-4 (40 mg) was purified further by
preparative TLC (CH2Cl2-EtOAc, 60:1) to obtain 3 (2.0 mg) (Rf )0.78)
and 4 (2.7 mg) (Rf ) 0.65). Fraction A6-5 (55 mg) was purified further
by preparative TLC (CHCl3-EtOAc, 10:1) to obtain 2 (4.7 mg) (Rf )
0.72). Fractions A8 and A9 (1.2 g) were chromatographed further on
silica gel (230-400 mesh, 100 g) eluting with n-hexane-acetone (3:
1) to give seven fractions (each 1.2 L, A8-1-A8-7). Fraction A8-3
(145 mg) was purified further by preparative TLC (n-hexane-EtOAc,
5:1) to obtain 13 (15.8 mg) (Rf ) 0.36). Fraction A8-5 (125 mg) was
purified further by preparative TLC (n-hexane-EtOAc, 3:1) to obtain
6 (12.4 mg) (Rf ) 0.55). Fraction A13 (0.9 g) was chromatographed
further on silica gel (230-400 mesh, 100 g) eluting with CHCl3-
acetone (10:1) to give eight fractions (each 1.2 L, A13-1-A13-8).
Fraction A13-5 (185 mg) was purified further by preparative TLC (n-
hexane-acetone, 5:1) to obtain 9 (11.4 mg) (Rf ) 0.58). Fraction A14
(0.8 g) was chromatographed further on silica gel (230-400 mesh, 90
g) eluting with CH2Cl2-acetone (5:1) to give seven fractions (each 1
L, A14-1- A14-7). Fraction A14-4 (135 mg) was purified further by
preparative TLC (n-hexane-EtOAc, 1:1) to obtain 12 (7.4 mg) (Rf )
0.60). Fraction A14-6 (128 mg) was purified further by preparative
TLC (CHCl3-acetone, 2:1) to obtain 11 (6.7 mg) (Rf ) 0.72). Fraction
A15 (0.75 g) was chromatographed further on silica gel (230-400
ibuprofenb
27.5 ( 3.45
a The IC50 values were calculated from the slope of the dose-
response curves. Values are expressed as the SEM of three independent
experiments. b Ibuprofen was used as a positive control.
carbonate in the biogenetic process. Compound 4 is a meso
25
compound, [R]D ) (0; thus 4 possessed a 4S,5R-configuration.
According to the above data, the structure of 4 was elucidated as
(4S,5R)-4,5-dimethyl-4,5-bis[(3,4,6-trimethoxy-2-methylphenyl)-
ethynyl]-1,3-dioxolan-2-one, named antrodioxolanone. This was
confirmed by 1H-1H COSY and NOESY (Figure 4) experiments.
The assignment of 13C NMR resonances was confirmed by DEPT,
HSQC, and HMBC (Figure 4) techniques.
The known isolates were readily identified by comparison of
physical and spectroscopic data (UV, IR, 1H NMR, [R]D, and MS)
with corresponding authentic samples or literature values, and this
included a benzenoid, 4,7-dimethoxy-5-methyl-1,3-benzodioxole
(5),5 a biphenyl, 2,2′,5,5′-tetramethoxy-3,4,3′,4′-bimethylenedioxy-
6,6′-dimethylbiphenyl (6),5 a lignan, (-)-sesamin (7),17 an R-to-
copheroid, R-tocospiro B (8),18 three triterpenoids, epi-friedelinol
(9),19 antcin A (10),3 antcin B (11),3 a steroidal acid, dehydroebu-
ricoic acid (12),7 and five steroids, ergosta-4,6,8(14),22-tetraen-3-
one (13),20 eburicol (14),21 â-sitostenone (15),22 and a mixture of
â-sitosterol (16)23 and stigmasterol (17).23
The anti-inflammatory effects of the isolates from the fruiting
body of A. camphorata were evaluated by suppressing N-formyl-
methionyl-leucyl-phenylalanine (fMLP)-induced production of su-
peroxide anion, an inflammatory mediator produced by neutrophils.
The anti-inflammatory activity data are shown in Table 1. The
clinically used anti-inflammatory agent ibuprofen was used as the
positive control. From the results of our anti-inflammatory tests,
the following conclusions can be drawn: (a) Antrocamphin A (1),
4,7-dimethoxy-5-methyl-1,3-benzodioxole (5), antcin A (10), and
antcin B (11) exhibited more potent inhibition (IC50 e 26.09 µM)
than ibuprofen (IC50 ) 27.52 µM) against fMLP-induced superoxide
production. (b) Antrocamphin A (1), with a 3-methylbut-3-en-1-
ynyl group, showed strong inhibition against fMLP-induced su-
peroxide production, but its analogue, antrocamphin B (2), with a
3-oxobut-1-ynyl group, was inactive. (c) 4,7-Dimethoxy-5-methyl-
1,3-benzodioxole (5) exhibited more effective inhibition than its
dimer, 2,2′,5,5′-tetramethoxy-3,4,3′,4′-bimethylenedioxy-6,6′-di-
methylbiphenyl (6), against fMLP-induced superoxide production.
(d) Antcin A (10) is the most effective among the isolates, with an
IC50 value of 8.55 ( 1.04 µM against fMLP-induced production
of superoxide anion by neutrophils. (e) Compounds 1, 5, 10, and
11 may exert their anti-inflammatory action through inhibiting
superoxide generation.

992 Journal of Natural Products, 2007, Vol. 70, No. 6
Chen et al.
mesh, 90 g) eluting with CH2Cl2-acetone (3:1) to give six fractions
(each 1 L, A15-1-A15-6). Fraction A15-5 (128 mg) was purified
further by preparative TLC (CH2Cl2-MeOH, 20:1) to obtain 10 (6.9
mg) (Rf ) 0.55).
Antrodioxolanone (4): yellowish, amorphous solid; [R]25 (0 (c
D
0.10, CDCl3); UV (MeOH) λmax (log ꢀ) 285 (4.15), 313 (4.08) nm; IR
(KBr) νmax 2186 (CtC), 1765 (CdO), 1598, 1497, 1455 (aromatic
ring CdC stretch) cm-1; 1H NMR (400 MHz) δ 1.70 (6H, s, Me-4 and
Me-5), 2.36 (6H, s, Me-2′ and Me-2′′), 3.72 (6H, s, OMe-3′ and Me-
3′′), 3.85 (6H, s, OMe-4′ and OMe-4′′), 3.87 (6H, s, OMe-6′ and OMe-
6′′), 6.33 (2H, s, H-5′ and H-5′′); 13C NMR (100 MHz) δ 14.2 (Me-2′
and Me-2′′), 20.1 (Me-4 and Me-5), 55.8 (OMe-6′ and OMe-6′′), 56.4
(OMe-4′ and OMe-4′′), 60.5 (OMe-3′ and OMe-3′′), 81.9 (C-4 and
C-5), 82.8 (Ph-CtC-4 and Ph-CtC-5), 94.2 (C-5′ and C-5′′), 92.6
(Ph-CtC-4 and Ph-CtC-5), 102.2 (C-1′ and C-1′′), 135.8 (C-2′ and
C-2′′), 141.3 (C-3′ and C-3′′), 152.5 (C-2), 154.0 (C-6′ and C-6′′), 158.5
(C-4′ and C-4′′); EIMS m/z (rel int) 524 (M+, 8), 509 (4), 369 (9), 355
(16), 295 (11), 281 (18), 246 (63), 231 (16), 221 (40), 209 (100), 207
(21), 203 (8), 165 (7), 129 (10), 111 (13), 98 (15); HRESIMS m/z
547.1946 [M + Na]+ (calcd for C29H32O9Na, 547.1944).
•-
Anti-inflammatory Activity Assay: Evaluation of O2 Release
by Human Neutrophils. Superoxide anion production was tested by
using a continuous spectrophotometric assay of ferricytochrome c
reduction by isolated neutrophils. Briefly, neutrophils were isolated from
the venous blood24 of consenting healthy volunteers (20-35 years old)
by double-gradient Ficoll-Hypaque centrifugation and hypotonic lysis
of contaminating red blood cells as previously described.25 Neutrophils
(1 × 106 cells/mL) pretreated with the various test agents (100 µmol/
L) at 37 °C for 5 min were stimulated with fMLP (1 µmol/L) in the
presence of ferricytochrome c (0.5 mg/mL). Extracellular O2•- produc-
tion was assessed with a UV spectrophotometer at 550 nm (Hitachi;
UV-3010). The percentage of superoxide inhibition of the test
compound was calculated as the percentage of inhibition ) {(control
- resting) - (compound - resting)}/(control - resting) × 100.
Antrocamphin A (1): yellowish oil; UV (MeOH) λmax (log ꢀ) 268
(4.12), 284 (4.23), 308 (4.13) nm; IR (KBr) νmax 2196 (CtC), 1593,
1490, 1462 (aromatic ring CdC stretch) cm-1; 1H NMR (400 MHz) δ
2.02 (3H, s, H-3′), 2.36 (3H, s, Me-3), 3.72 (3H, s, OMe-2), 3.87 (3H,
s, OMe-1), 3.88 (3H, s, OMe-5), 5.25 (1H, br s, Ha-4′), 5.37 (1H, br
s, Hb-4′), 6.33 (1H, s, H-6); 13C NMR (100 MHz) δ 14.1 (Me-3), 23.7
(Me-3′), 55.8 (OMe-5), 56.3 (OMe-1), 60.4 (OMe-2), 83.6 (C-1′), 94.3
(C-6), 97.5 (C-2′), 104.8 (C-4), 120.7 (C-4′), 127.3 (C-3′), 135.3 (C-
3), 141.1 (C-2), 153.5 (C-5), 157.2 (C-1); EIMS m/z (rel int) 246 ([M]+,
73), 231 (50), 203 (72), 188 (40), 173 (27), 160 (28), 145 (34), 129
(33), 128 (47), 127 (23), 117 (55), 115 (82), 91 (32), 89 (20), 77 (43),
73 (32), 69 (100), 65 (25), 63 (44), 57 (23), 55 (37), 51 (43); HRESIMS
m/z 247.1337 [M + H]+ (calcd for C15H19O3, 247.1334).
Acknowledgment. This work was supported by a grant from the
National Science Council of the Republic of China.
References and Notes
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1389-1392.
Antrocamphin B (2): yellowish powder; UV (MeOH) λmax (log ꢀ)
295 (3.98), 337 (4.07) nm; IR (KBr) νmax 2182 (CtC), 1661 (CdO),
(8) Cherng, I. H.; Wu, D. P.; Chiang, H. C. Phytochemistry 1996, 41,
263-267.
1
1591, 1488, 1462 (aromatic ring CdC stretch) cm-1; H NMR (400
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MHz) δ 2.46 (3H, s, H-4′), 2.39 (3H, s, Me-3), 3.73 (3H, s, OMe-2),
3.89 (3H, s, OMe-1), 3.92 (3H, s, OMe-5), 6.32 (1H, s, H-6); 13C NMR
(100 MHz) δ 14.3 (Me-3), 33.0 (C-4′), 56.1 (OMe-5), 56.4 (OMe-1),
60.7 (OMe-2), 88.1 (C-1′), 94.0 (C-6), 96.5 (C-2′), 101.5 (C-4), 137.6
(C-3), 141.4 (C-2), 156.4 (C-5), 160.0 (C-1), 184.8 (C-3′); EIMS m/z
(rel int) 248 ([M]+, 98), 233 (77), 175 (31), 161 (29), 153 (31), 147
(23), 136 (26), 107 (34), 105 (20), 91 (24), 89 (48), 78 (35), 77 (100),
76 (28), 74 (23), 69 (62), 65 (22), 63 (58); HRESIMS m/z 249.1128
[M + H]+ (calcd for C14H17O4, 249.1127).
(10) Shen, C. C.; Kuo, Y. C.; Huang, R. L.; Lin, L. C.; Don, M. J.; Chang,
T. T.; Chou, C. J. J. Chin. Med. 2003, 14, 247-258.
(11) Nakamura, N.; Hirakawa, A.; Gao, J. J.; Kakuda, H.; Shiro, M.;
Komatsu, Y.; Sheu, C. C.; Hattori, M. J. Nat. Prod. 2004, 67, 46-
48.
(12) Shen, Y. C.; Wang, Y. H.; Chou, Y. C.; Chen, C. F.; Lin, L. C.;
Chang, T. T.; Tien, J. H.; Chou, C. J. Planta Med. 2004, 70, 310-
314.
(13) Chen, C. C.; Shiao, Y. J.; Lin, R. D.; Shao, Y. Y.; Lai, M. N.; Lin,
C. C.; Ng, L. T.; Kuo, Y. H. J. Nat. Prod. 2006, 69, 689-691.
(14) Meyers, A. I.; Flisak, J. R.; Aitken, R. A. J. Am. Chem. Soc. 1987,
109, 5446-5452.
2,3,4,5-Tetramethoxybenzoyl chloride (3): amorphous powder; UV
(MeOH) λmax (log ꢀ) 210 (4.59), 245 (4.08), 300 (3.69) nm; IR (KBr)
1
νmax 1768 (CdO) cm-1; H NMR (400 MHz) δ 3.89 (3H, s, OMe-4),
3.91 (3H, s, OMe-5), 3.92 (3H, s, OMe-3), 3.93 (3H, s, OMe-2), 7.13
(1H, s, H-6); 13C NMR (100 MHz) δ 56.3 (OMe-5), 61.1 (OMe-4),
61.2 (OMe-3), 62.4 (OMe-2), 109.7 (C-6), 116.5 (C-1), 151.0 (C-4),
146.5 (C-5), 147.9 (C-2), 148.2 (C-3), 168.1 (CdO); EIMS m/z (rel
int) 260 ([M]+, 100), 245 (64), 231 (16), 229 (54), 227 (39), 217 (56),
219 (19), 213 (32), 202 (25), 173 (21), 171 (54), 143 (21), 131 (25),
129 (32), 105 (32), 103 (35), 84 (48), 77 (81); HRESIMS m/z 283.0350
[M + Na]+ (calcd for C11H13ClO5Na, 283.0349).
(15) Ghosal, S.; Chaudhuri, R. K. J. Chem. Soc., Perkin Trans. 1 1974,
2538-2541.
(16) Lu, L.; Fang, J. M.; Lee, G. H.; Wang, Y. J. Chin. Chem. Soc. 1997,
44, 279-289.
(17) Yu, H. J.; Chen, C. C.; Shieh, B. J. J Chin. Chem. Soc. 1998, 45,
773-778.
(18) Chiang, Y. M.; Kuo, Y. H. Tetrahedron Lett. 2003, 44, 5125-5128.
(19) Salazer, G. C. M.; Silva, G. D. F.; Duarte, L. P.; Vieira, F. S. A.;
Lula, I. S. Magn. Reson. Chem. 2000, 38, 977-980
(20) Kobayashi, M.; Krishna, M. M.; Ishida, K.; Anjanetulu, V. Chem.
Pharm. Bull. 1992, 40, 72-74.
Methyl 2,3,4,5-tetramethoxybenzoate (3a). Methanol (0.3 mL) was
added to a solution of 3 (1.9 mg) in pyridine (0.3 mL), and the mixture
was stirred for 10 h at room temperature. The excess pyridine was
removed with saturated aqueous CuSO4 solution to give a residue (1.8
mg). The residue was chromatographed on silica gel to give 3a (1.5
mg), colorless oil: UV (MeOH) λmax (log ꢀ) 209 (4.50), 243 (4.04),
306 (3.61) nm; IR (KBr) νmax 1714 (CdO) cm-1; 1H NMR (400 MHz)
δ 3.89 (3H, s, OMe-4), 3.90 (3H, s, COOMe), 3.90 (3H, s, OMe-5),
3.91 (3H, s, OMe-3), 3.91 (3H, s, OMe-2), 7.03 (1H, s, H-6); EIMS
m/z (rel int) 256 ([M]+, 100), 241 (54), 225 (51), 210 (57), 194 (14),
171 (55), 131 (27), 129 (29), 103 (31), 84 (42), 77 (78); HREIMS m/z
256.0942 [M]+ (calcd for C12H16O6, 256.0947).
(21) Shirane, N.; Takenaka, H.; Ueda, K.; Hashimoto, Y.; Katoh, K.; Ishii,
H. Phytochemistry 1996, 41, 1301-1308.
(22) Betancor, C.; Freire, R.; Gonzalez, A. G.; Salazar, J. A.; Pascard,
C.; Prange, T. Phytochemistry 1980, 19, 1989-1993.
(23) Chen, J. J.; Huang, Y. C.; Chen, Y. C.; Huang, Y. T.; Wang, S. W.;
Peng, C. Y.; Teng, C. M, Chen, I. S. Planta Med. 2002, 68, 980-
985.
(24) Boyum, A. Scand. J. Lab. InVest. 1968, 21, 77-97.
(25) English, D.; Andersen, B. R. J. Immunol. Methods 1974, 5, 249-252.
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