First total synthesis of antrocamphin A and its analogs as anti-inflammatory and anti-platelet aggregation agents

Article abstract of DOI:10.1039/c0ob00616e

Naturally occurring antrocamphin A (1) is a potent anti-inflammatory compound from the edible fungus Antrodia camphorata (Taiwanofungus camphoratus), whose wild fruiting body is used as a valuable folk medicine in Taiwan. This study is the first total syn

Full text of DOI:10.1039/c0ob00616e

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First total synthesis of antrocamphin A and its analogs as anti-inflammatory
and anti-platelet aggregation agents†
Chia-Lin Lee,a,h Chi-Huan Huang,‡a Hui-Chun Wang,a Da-Wei Chuang,a Ming-Jung Wu,b Sheng-Yang Wang,c
Tsong-Long Hwang,d Chin-Chung Wu,a Yeh-Long Chen,e Fang-Rong Chang*a, f and Yang-Chang Wu*a,g,h
Received 23rd August 2010, Accepted 1st November 2010
DOI: 10.1039/c0ob00616e
Naturally occurring antrocamphin A (1) is a potent anti-
inflammatory compound from the edible fungus Antrodia
camphorata (Taiwanofungus camphoratus), whose wild fruit-
ing body is used as a valuable folk medicine in Taiwan. This
study is the first total synthesis of antrocamphin A (1) and its
analogs. Their inhibition ability on NO release, superoxide
anion generation, elastase release and platelet aggregation
Fig. 1 Antrocamphin A (1).
are reported herein.
shown below through a complete bioassay-guided fractionation
study.3,4
Antrocamphin A (1), a potent anti-inflammatory compound
naturally found in A. camphorata (T. camphoratus) and was first
Introduction
isolated by Chen et al. in 2007.4 Wang et al. demonstrated its
mechanism in suppressing pro-inflammatory molecules (NO and
PGE2), from being released via the down-regulation of iNOS and
COX-2 expression through the NF-kB pathway.3 Previous studies
indicate that compound 1 may serve as a promising lead drug for
the treatment of various diseases that are induced by inflammation.
Therefore, antrocamphin A was chosen to be a candidate of further
investigation.
Currently, there are only two literature3,4 reports on the subject
of antrocamphin A (1). To understand the diverse biological
properties and the structure–activity relationship (SAR) of the
lead compound 1, the first total synthesis of compound 1 and its
analogs were achieved in the current investigation. All compounds
were evaluated for anti-inflammatory and anti-platelet aggrega-
tion activities.
The endemic fungus Antrodia camphorata (Taiwanofungus cam-
phoratus), also known as Niu-Chang-Chih (Jang-Jy), is used as a
folk medicine and a dietary supplement in Taiwan. Its wild fruiting
body is very valuable.1–3 The chemical composition of this fungus
can be classified into three categories: 1). Polysaccharides, 2).
Triterpenoids and 3). Enynyl-benzenoids. The polysaccharides are
regarded as immunomodulation agents, such as functional foods
derived from other fungi, e.g., Ganoderma lucidum and Agaricus
blazei.1 Triterpenoids are considered to have anti-cancer and anti-
inflammatory effects. However, the major chemical component,
antrocamphin A (1) (Fig. 1), which belongs to the third class,
is the key component for anti-inflammatory activity. Evidence is
aGraduate Institute of Natural Products, Kaohsiung Medical University,
Kaohsiung 807, Taiwan. E-mail: aaronfrc@kmu.edu.tw; Fax: +886 7 311
4773; Tel: +886 7 312 1101 ext. 2162
bDepartment of Chemistry, National Sun Yant-sen University, Kaohsiung
804, Taiwan
Results and discussion
cDepartment of Forestry, National Chung-Hsing University, Taichung 402,
Taiwan
The retrosynthetic analysis of compound 1 is illustrated in Scheme
1. The key step was the Sonogashira reaction. Compound 2
(2-iodo-3,5,6-trimethoxytoluene) was coupled with 2-methyl-1-
buten-3-yne (3), which led to the target compound. Compound 2
was synthesized from 2,3,5-trimethoxytoluene (8), obtained from
o-vanillin (5), in four steps described by Singh and co-workers.5
We prepared 2-hydroxy-3-methoxytoluene (4) by hydrogenating of
o-vanillin (5) over 10% palladium-on-carbon. Compound 4 was
then converted to quinone 6 by treatment with the oxidant, potas-
sium nitrosodisulfonate (Fremy’s salt). Quinone 6 was reduced to
hydroquinone 7 using TiCl3 and then methylated with Me2SO4
in the presence of K2CO3 resulting in the desired compound 8.
Compound (2), 2-iodo-3,5,6-trimethoxytoluene, a key synthon,
dGraduate Institute of Natural Products, Chang Gung University, Tao-Yuan
333, Taiwan
eDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical
University, Kaohsiung 807, Taiwan
f
Department of Marine Biotechnology and Resources, National Sun Yat-sen
University, Kaohsiung 804, Taiwan
gGraduate Institute of Integrated Medicine, College of Chinese Medicine,
China Medical University, Taichung 40402, Taiwan. E-mail: yachwu@
mail.cmu.edu.tw; Fax: +886 4 220 60248; Tel: +886 4 220 57153
hNatural Medicinal Products Research Center, China Medical University
Hospital, Taichung 40402, Taiwan
† Electronic supplementary information (ESI) available: Experimental
details and spectral data. See DOI: 10.1039/c0ob00616e
‡ Equal contributions as first author.
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Scheme 1 Retrosynthetic analysis of antrocamphin A.
was prepared via iodination of 8 using I2 and CF3COOAg. The
Sonogashira reaction was utilized to couple iodo 2 with 2-methyl-
1-buten-3-yne (3), using the catalysts Pd(PPh3)4 and CuI. This
key reaction led to the desired compound, antrocamphin A (1)
(Scheme 2).6 The Sonogashira coupling reaction was also applied
to afford a series of designed analogues (9–22) from commercially
available iodobenzene products or further iodination benzenoid
compounds (Scheme 3).
Synthesized compounds 1 and 9–22 were screened using a
nitric oxide (NO) inhibitory assay, where curcumin was used
as the positive control. In this study, all analogs were tested
on their cytotoxicity toward RAW 264.7 macrophages. Cell
viability rates for each test compound showed > 90% at the
dosage of 20 mg mL-1. All compounds were evaluated against
lipopolysaccharide (LPS)-induced NO production in RAW 264.7
macrophage cell line at the dosage of 20 mg mL-1. As shown in
Table 1, compounds 1, 20, 21 and 22 showed potent anti-
inflammatory activity with NO inhibition rates of 98.1%, 97%,
77%, 84%, respectively.
Compounds 1, 9, 15, 17, 20 and 21 were synthesized to study
the SAR of the CH3 substitution at C-3 presented in the natural
product 1; however, C3-CH3 did not influence the ability of NO
inhibition. To evaluate the SAR of the OCH3 functions on the 3¢-
methyl-but-3¢-en-1-ynyl-benzene skeleton, analogs 10–12, 15 and
Scheme
3
Synthesis of 9–22. Reagents and conditions: (a)
2-methyl-1-buten-3-yne (3), Pd(PPh3)4, CuI, Et3N/THF, N2, rt.
20 were designed, which did not have the C3-CH3 substitution.
Compounds 10–12 possess only one OCH3 group at C-1, C-3
and C-2, respectively. Moreover, 20 and 15 have two and three
OCH3 groups at C-1 and C-5, and C-1, C-2 and C-5, respectively.
Among the five compounds, we speculated that the position is
more important than the number of OCH3 substitutions. A similar
result was also observed in the test of compounds 19 and 21, which
both have a CH3 group at C-3 and the same number of OCH3
substitutions in different positions. Furthermore, a comparison
Scheme 2 Synthetic scheme of antrocamphin A (1). Reagents and conditions: (a) 10% Pd/C, H2, rt, 75%; (b) KH2PO4, (KSO3)2NO, rt, 78%; (c) TiCl3, rt,
90%; (d) (CH3O)2SO2, K2CO3, acetone, reflux, 88%; (e) I2, CF3COOAg, CH2Cl2, 0 ◦C, 74%; (f) 2-methyl-1-buten-3-yne (3), Pd(PPh3)4, CuI, Et3N/THF,
N2, rt, 10%.
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Table 1 Effects of all compounds on NO in LPS-challenged RAW 264.7
Table 2 Effects of all compounds on superoxide anion generation and
elastase release by human neutrophils in response to FMLP/CB
cellsa
Compoundb
NO inhibition (%)
Cell viability (%)
Superoxide
Elastase
Compound IC50/mg mL-1a or (Inh %) IC50/mg mL-1a or (Inh %)
1
98.10
17.23
33.56
-4.43
27.27
23.11
-0.55
24.98
15.16
6.23
16.61
28.51
97.00
77.00
84.00
99.00
99.71
9
101.55
101.98
90.44
105.80
100.95
91.77
101.91
94.97
99.73
92.23
99.07
117.08
114.00
102.00
103.00
1
(46.30 7.24)
4.82 0.41
5.63 0.71
6.69 0.84
4.05 0.41
4.66 0.41
(7.95 6.06)
3.40 1.49
(-10.03 4.84)
(25.55 6.70)
0.45 0.03
1.96 0.28
4.69 0.99
1.60 0.28
(12.13 2.24)
0.22 0.13
***
(25.55 3.99) **
(37.22 3.12) ***
4.14 0.35
10
9
11
10
12
11
7.94 0.51
13
12
3.31 0.06
44.28 1.67
14
13
***
15
14
(17.89 1.42) ***
16
15
(26.74 5.88)
22.78 1.01
*
***
17
16
18
17
*
(35.55 6.12) **
1.32 0.67
3.47 0.23
8.72 1.15
5.33 1.21
19
18
20
19
21
20
22
Curcuminc
21
22
**
(81.36 8.82)
DPIb
PMSFb
a All compounds were administrated 1 h before inflammation induction
by adding LPS (1 mg mL-1). b Dosage of test compound was 20 mg mL-1.
c Positive control.
22.80 5.07
Per cent of inhibition (Inh%) at a 10-mg mL-1 concentration. Results are
presented as mean S.E.M. (n = 3). *p < 0.05, **p < 0.01, ***p <
0.001 compared with the control value.a Concentration necessary for 50%
inhibition (IC50). b Diphenyleneiodonium (DPI) and phenylmethylsulfonyl
fluoride (PMSF) were used as positive controls for superoxide anion
generation and elastase release, respectively.
with the structures of 1 and 20–22 concluded that the simultaneous
existence of OCH3 groups at C-1 and C-5 may be necessary for the
potential ability of NO inhibition.
LPS-challenged RAW 264.7 macrophages are an in vitro model
of murine cell lines for studying anti-inflammatory effects. To
further explore the anti-inflammatory activities of these target
compounds in human cell systems with different modes of action,
all analogs were evaluated against superoxide anion genera-
tion and elastase release by human neutrophils in response to
N-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin
Table 3 Anti-platelet aggregation data of all compounds
IC50/mg mL-1
Compound
Collagen (10 mg mL-1)
Thrombin (0.05 U/mL)
1
>50
3.36
1.16
7.55
>50
>50
12.14
15.43
>50
>50
14.50
7.22
>50
>50
>50
13.58
>50
40.83
44.06
>50
>50
>50
36.09
>50
>50
>50
12.92
>50
>50
>50
>50
>200
B
(CB). Antrocamphin A has been reported as a new
9
anti-inflammatory drug lead against superoxide anion generation
in FMLP-activated human neutrophils.4 Activated neutrophils
produced high concentrations of the superoxide anion and elastase
known to be involved in airway mucus hypersecretion, which is
a neutrophilic inflammatory disease like asthma.7 Therefore, all
analogues were tested for their ability to inhibit superoxide anion
generation and elastase release. Diphenyleneiodonium (DPI) and
phenylmethylsulfonyl fluoride (PMSF) were used as positive
controls in this model assay, respectively.
Interestingly, the analogs showed a broad-spectrum of activity
in this model (Table 2). Most of the compounds could inhibit both
inflammatory mediators and showed more potent effects than the
natural product 1. Compound 18 with a dienynyl functionality
exhibited the best activity, which indicated the enynyl substitution
played an important role for this model assay. On the whole, we
found dimethoxy (19–22) and monomethoxy (10–12) derivatives
were more favorable than trimethoxy and non-methoxy ones.
Some of the compounds showed superior activities to the reference
drug, PMSF, against elastase release by neutrophils.
10
11
12
13
14
15
16
17
18
19
20
21
22
Aspirina
a Positive control.
bioassay results, most of the compounds did not possess good
inhibition toward the platelet aggregation induced by thrombin.
No clear SAR can be concluded from the anti-platelet aggregation
effect. Interestingly, compounds 1 and 20–22, which possess the
powerful ability of NO inhibition, did not have anti-platelet
aggregation activity.
Furthermore, all synthesized derivatives were also subjected to
an anti-platelet aggregation assay with collagen and thrombin as
inducers; aspirin was used as a positive control. As shown in Table
3, compounds 9, 10, 11 and 19 showed potent inhibition against
collagen-induced platelet aggregation with IC50 values of 3.36,
1.16, 7.55 and 7.22 mg mL-1, respectively; their activities were
better than aspirin’s activity (IC50 13.58 mg mL-1). Comparing the
Conclusions
In summary, we have achieved the first total synthesis of the
active natural product antrocamphin A (1), in a total yield
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of 3.7% in six steps. Moreover, 14 analogs, including new
compounds, 15, 16 and 18–22, were synthesized. Most of these
compounds possess OMe groups in the benzene ring. We tried
to synthesized derivatives with different substitutions around the
ring, for example, 2-iodo-3,6-diacetoxy-5-methoxytoluene or 4-
iodo-3,6-dihydroxy-5-methoxytoluene coupled with 2-methyl-1-
buten-3-yne using different combinations of catalyst [Pd(PPh3)4 or
PdCl2(PPh3)2], solvent (THF or DMF or ether) and base (Et3N).
Unfortunately, this resulted in no reaction or reaction without
any target compound. Accordingly, only the reagents with OMe
groups, which were easily obtained, were chosen to evaluate the
amount and position of OMe groups related to the activity in this
investigation.
Comparing anti-inflammatory activity, compounds 1, 20, 21
and 22 were identified to be potent NO inhibition agents; the
dienynyl compound 18 was a new drug lead against superoxide
anion generation and elastase release. Moreover, analog 10 is the
most potential compound against platelet aggregation induced by
collagen. Overall, our data demonstrate that enynyl-benzenoids
may have a chance to be developed as safe and potential
anti-inflammatory or a cardiovascular protecting agent in the
future.
Acknowledgements
This work was supported by grants from National Science
Council, Taiwan awarded to Y.-C. Wu and F.-R. Chang. This
study is also supported in part by Taiwan Department of Health
Clinical Trial and Research Center of Excellence (DOH99-TD-B-
111-004) and Department of Health Cancer Research Center of
Excellence (DOH99-TD-C-111-005).
Notes and references
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