An efficient total synthesis of Benzocamphorin H and its anti-inflammatory activity

Article abstract of DOI:10.1016/j.tetlet.2012.08.138

A naturally occurring enynyl-benzenoid, Benzocamphorin H (1), from the edible fungus Taiwanofungus camphoratus (Antrodia camphorata) was characterized by the comprehensive spectral analysis. It displayed potent anti-inflammatory bioactivity and would be v

Full text of DOI:10.1016/j.tetlet.2012.08.138

Tetrahedron Letters 53 (2012) 6202–6204
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An efficient total synthesis of Benzocamphorin H and its
anti-inflammatory activity
Yu-Ren Liao a, , Ping-Chung Kuo b, Shiow-Chyn Huang c, , Jun-Weil Liang a, Tian-Shung Wu a,d,e,
⇑
a Department of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
b Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, ROC
c Graduate Institute of Pharmaceutical Science, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan, ROC
d Department of Pharmacy, China Medical University, Taichung 404, Taiwan, ROC
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 404, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2012
Revised 22 August 2012
Accepted 30 August 2012
Available online 7 September 2012
A naturally occurring enynyl-benzenoid, Benzocamphorin H (1), from the edible fungus Taiwanofungus
camphoratus (Antrodia camphorata) was characterized by the comprehensive spectral analysis. It dis-
played potent anti-inflammatory bioactivity and would be valuable for further biological studies. The
present study is the first total synthesis of Benzocamphorin H and the developed strategy described a
more efficient procedure that allowed large-scale production of Benzocamphorin H for the further
research of biological activity both in vitro and in vivo.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Antrodia camphorata
Benzocamphorin H
Anti-inflammatory
Taiwanofungus camphoratus (synonyms: Ganoderma camphora-
tum; Antrodia cinnamomea; Antrodia camphorata) (Polyporaceae,
Aphyllophorales) is a rare and precious medical fungus in Taiwan
and is called a ‘national treasure of Taiwan’ with the Chinese name
as Zhan-Ku or Niu-Chang-Chih.1 The microorganism is parasitic to
the inner heartwood wall of old hollow trunks of Cinnamomum
kanehirai Hay (Lauraceae). In traditional Taiwanese folk medicine
T. camphoratus has been used as an important health food for treat-
ing food, alcohol, and drug intoxication, diarrhea, abdominal pain,
hypertension, itching, and liver cancer.2,3 Previous studies have re-
vealed that Niu-Chang-Chih exerts various biological activities,
such as hepatoprotective, antihepatitis B virus, anticancer, antiox-
idant, and anti-inflammatory activities.4,5 The chemical constitu-
ents of this fungus could be divided into three classes, including
polysaccharides, triterpenoids, and enynyl-benzenoids.6,7 Our
ongoing study on the chemical composition of the ethanol extract
of the fruiting body of T. camphoratus has led to the isolation of one
new enynyl-benzenoid, Benzocamphorin H (1) (Fig. 1).
inducible NO synthase (iNOS) in leukocytes, fibroblasts, and other
cell types, accounting for enhanced levels of NO. Although NO is a
microbicide and may have important roles in tissue adapting to
inflammatory states, overproduction of NO may exacerbate tissue
injury in both acute and chronic inflammatory conditions. In the
experimental model of acute inflammation, inhibition of iNOS
can have a dose-dependent protective effect, suggesting that NO
promotes edema and vascular permeability. NO also has a detri-
mental effect in chronic models of arthritis, whereas protection is
seen with iNOS inhibitors. Glucocorticoids, which are often used
in the treatment of inflammation, are able to inhibit the expression
of iNOS. Therefore, the extracts of T. camphoratus which were con-
sidered as the rich sources of triterpenoids were examined for their
anti-inflammatory activity to explore the new lead drugs. In the
present study, we wish to report the structural determination of
one new enynyl-benzenoid from T. camphoratus, as well as evalu-
ation of its anti-inflammatory activity. In addition, we developed
a
more efficient synthetic protocol that allowed large-scale
Inflammation, which is related to morbidity and mortality of
many diseases, is part of the complex biological response of vascu-
lar tissues to harmful stimuli, and is the host response to infection
or injury, which involves the recruitment of leukocytes and the re-
lease of inflammatory mediators, including nitric oxide (NO). NO is
the metabolic by-product of the conversion of transcription of
4'
4'
CH3
2'
CH3
2'
3'
3'
H3C
1'
H3C
1'
3
3
2
OH
2
OCH3
OCH3
4
4
1
1
5
5
H3CO
OCH3
H3CO
6
6
⇑
2
Corresponding author. Tel.: +886 6 275 7575; fax: +886 6 274 0552.
E-mail address: tswu@mail.ncku.edu.tw (T.-S. Wu).
1
These authors had equal contributions to this work.
Figure 1. Structures of Benzocamphorin H (1) and Antrocamphin A (2).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.08.138

Y.-R. Liao et al. / Tetrahedron Letters 53 (2012) 6202–6204
6203
production of Benzocamphorin H for the further research of biolog-
ical activity.
Benzocamphorin H (1) was isolated as colorless powder and
CHO
CH3
H3C
OH
OH
showed a [M+Na]+ ion peak at m/z 255.0997 in its HRESIMS, corre-
sponding to the molecular formula C14H16O3Na. The UV spectrum
of 1 displayed absorption maxima at 256, 285, and 318 nm, and
the IR spectrum exhibited strong absorption peaks for hydroxy
group (3529 cmꢀ1), carbon–carbon triple bond (2197 cmꢀ1), and
carbon–carbon double bond (1605 cmꢀ1), respectively. In the 1H-
NMR spectrum, there were signals observed for a set of single aro-
matic protons at d 6.34 (1H, s, H-6), 5.31 (1H, br s, OH-2), terminal
methylene protons at d 5.38 (1H, s, H-40) and d 5.25 (1H, s, H-40),
two methoxy singlets at d 3.90 (3H, H-1) and 3.85 (3H, H-5), and
two methyl singlets at d 2.34 (3H, H-3) and 2.02 (3H, H-30), respec-
tively. The 13C NMR and DEPT spectra combined with HMQC exper-
iment indicated 14 signals including an olefinic carbon resonance
at d 120.7, two methoxy groups at d 56.8 and 56.0, one aromatic
methine at d 93.8, two methyl groups at d 23.7 and 13.6, seven qua-
ternary carbons at d 154.3, 146.6, 127.3, 126.8, 105.4, 97.5, and
83.6, and one oxygen-bearing quaternary carbon at d 137.6. The
HMBC correlations (Fig. 2) from OCH3-1 (d 3.90) to C-1 (d 146.6),
from OH-2 (d 5.31) to C-1 (d 146.6)/C-2 (d 137.6)/C-3 (d 126.8),
from CH3-3 (d 2.34) to C-2 (d 137.6)/C-3 (d 126.8)/C-4 (d 105.4),
from OCH3-5 (d 3.85) to C-5 (d 154.5), from H-6 (d 6.34) to C-1 (d
146.6)/C-2 (d 137.6)/C-4 (d 105.4)/C-5 (d 154.5), from CH3-30 (d
2.02) to C-20 (d 97.5)/C-30 (d 127.3)/C-40 (d 120.7), and from H-40
(d 5.38, 5.25) to C-20 (d 97.5)/C-30 (d 127.3)/CH3-30 (d 23.7) con-
structed the substituted pattern of this enynyl-benzenoid. On the
basis of these spectral data, the chemical structure of 1 was iden-
tified as shown in Figure 1. It is the first report of this compound
from the natural sources and it was given the trivial name, Benzoc-
amphorin H, proposed following a previous convention.
H3CO
OCH3
H3CO
OCH3
3
1
CHO
H3CO
OCH3
4
Figure 3. Retro-synthetic analysis of Benzocamphorin H (1).
hyde (6) was prepared via bromination of 3 using N-Bromosuccin-
imide and CH3COOH. Hydrogenation of 6 by over 10% palladium-
on-carbon reduced the formyl substituent to methyl group and
produced 2-bromo-3,5-dimethoxy-6-hydroxy toluene (7).11 Com-
pound 7 was coupled with 2-methyl-3-butyn-2-ol by Sonogashira
reaction, which successively led to compound 8.12 Finally the
dehydration of compound 8 in toluene by methanesulfonyl chlo-
ride was carried out to produce Benzocamphorin H (1) in good
yield (92%). The present synthetic protocol of Benzocamphorin H
(1) provides a more efficient synthetic pathway with satisfied over-
all yield (6 steps, 41.2%). The chemical structure of synthetic com-
pound
1 was elucidated by 1D and 2D-NMR and mass
spectrometry and compared with that of the purified natural com-
pound. In addition, the reagents used in this synthesis were com-
paratively of low cost, thus it would be more suitable for the
large-scale production of Benzocamphorin H and the analogs with
similar skeleton.
In summary, a new enynyl-benzenoid, Benzocamphorin H (1),
was isolated from the fruiting body of Taiwanofungus camphoratus
for the first time. The structure of 1 was fully elucidated by 2D-
NMR analysis and also by an efficient and simple method for chem-
ical synthesis. This novel method has several advantages over the
other methods, such as high yield, fewer reaction steps, faster reac-
tion rate, and easy work up without producing any significant by-
product. The bioactivity examination results indicated that com-
pound 1 displays potent NO-reducing activities in microglial cells
and thus it had the potential to be an anti-inflammatory drug for
the treatment of NO-dependent neurodegenerative disorders.
The inhibitory effects of Benzocamphorin H on LPS-induced NO
production in RAW 264.7 macrophages were also investigated. Ni-
trite accumulated in the culture medium was estimated by the Gri-
ess reaction as an index for NO release from the cells. When RAW
264.7 macrophages were treated with different concentrations of
Benzocamphorin H together with LPS (0.5 mg/mL) for 24 h, a sig-
nificant concentration-dependent inhibition of nitrite production
was detected. The IC50 value for inhibition of nitrite production
of Benzocamphorin H was 15.09 1.21 lM.
In the previous literature, Wu et al. reported a total synthesis of
Antrocamphin A (2) in six steps with an overall yield of 3.7%.8
However, the low yield and high cost of the reagents for this meth-
od would reduce the potential of application. Herein we wish to re-
port a more efficient and economic method to prepare the analogs
possessing the same skeleton as that of 2. The retro-synthetic anal-
ysis of Benzocamphorin H (1) is displayed in Figure 3 and thus we
initiated the preparation of 1 from 3,5-dimethoxybenzaldehyde
(4). The NBS-assisted bromination of 4 resulted in 2-bromo-3,5-
dimethoxybenzaldehyde (5) as shown in Scheme 1. Compound 5
was hydrolyzed in 8% (w/w) sodium hydroxide aqueous solution
in the presence of 3% (w/w) copper to afford the 2-hydroxy-3,5-
dimethoxybenzaldehyde (3) in 80% yield.9,10 Increasing the
percentages of base or metal would not improve the yields of the
hydrolytic reaction. 2-Bromo-3,5-dimethoxy-6-hydroxy-benzalde-
CHO
CHO
CHO
Br
OH
a
b
88%
80%
H3CO
OCH3
H3CO
OCH3
H3CO
OH
OCH3
4
5
3
CHO
CH3
e
c
Br
OH
Br
H3CO
d
86%
87%
85%
H3CO
OCH3
OCH3
6
7
HO
CH3
H
H
CH3
4'
H3C
f
OH
OCH3
OH
OCH3
CH3
2'
3'
H3C
1'
3
92%
OH
1 OCH3
2
H3CO
H3CO
4
8
1
5
H3CO
6
H
Scheme 1. Reagents and conditions: (a) NBS, acetonitrile, rt; (b) NaOH (aq), Cu,
reflux; (c) NBS, acetic acid, rt; (d) H2, Pd/C, MeOH, rt; (e) 2-methyl-3-butyn-2-ol,
Pd(PPh3)4, CuI, DMF; (f) methanesulfonyl chloride, toluene, microwave.
Figure 2. HMBC (¡ꢁ) correlations of 1.

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Y.-R. Liao et al. / Tetrahedron Letters 53 (2012) 6202–6204
2. Tsai, Z. T.; Liaw, S. L. The Use and the Effect of Ganoderma; Sang-Yun Press:
Acknowledgment
Taichung, Taiwan, 1982. p 116.
3. Chen, C. J.; Su, C. H.; Lan, M. H. Fungal Sci. 2001, 16, 65.
4. Liu, D. Z.; Liang, H. J.; Chen, C. H.; Su, C. H.; Lee, Z. H.; Huang, C. T.; Hou, W. C.;
Lin, S. Y.; Zhong, W. B.; Lin, P. J.; Hung, L. F.; Liang, Y. C. J. Ethnopharmacol. 2007,
113, 45.
This work was supported by a Grant of National Science Coun-
cil, Taiwan, Republic of China, awarded to T.S. Wu.
5. Rao, Y. K.; Fang, S. H.; Tzeng, Y. M. J. Ethnopharmacol. 2007, 114, 78.
6. Wu, S. J.; Leu, Y. L.; Chen, C. H.; Chao, C. H.; Shen, D. Y.; Chan, H. H.; Lee, E. J.;
Wu, T. S. J. Nat. Prod. 2011, 73, 1756.
Supplementary data
7. Shi, L. S.; Chao, C. H.; Shen, D. Y.; Chan, H. H.; Chen, C. H.; Wu, S. J.; Leu, Y. L.;
Shen, Y. C.; Kuo, Y. H.; Lee, E. J.; Wu, T. S.; Lee, K. H. Bioorg. Med. Chem. 2011, 19,
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Hwang, T. L.; Wu, C. C.; Chen, Y. L.; Chang, F. R.; Wu, Y. C. Org. Biomol. Chem.
2011, 9, 70.
Supplementary data (general experimental information and
experimental detail) associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
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