Reprint of: Antiproliferative activity of the Antrodia camphorata secondary metabolite 4,7-dimethoxy-5-methylbenzo[d][1,3]dioxole and analogues

Article abstract of DOI:10.1016/j.fitote.2018.04.001

Both the traditional Chinese medicinal fungus, Antrodia camphorata, and its secondary metabolite, 4,7-dimethoxy-5-methylbenzo[d][1,3]dioxole, have been reported to possess promising anticancer activity. In this work the natural product and analogues bearing more polar substituents were synthesised and assessed for antiproliferative activity in the NCI-60 screen. Although each compound inhibited the growth of some cell lines at 10 μM, none had sufficient activity to warrant further investigation.

Full text of DOI:10.1016/j.fitote.2018.04.001

Fitoterapiaxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Reprint of: Antiproliferative activity of the Antrodia camphorata secondary
metabolite 4,7-dimethoxy-5-methylbenzo[d][1,3]dioxole and analogues☆
Sing Yee Yeung, Matthew J. Piggott⁎
Chemistry, School of Molecular Sciences, University of Western Australia, Perth, 6009, Australia
A R T I C L E I N F O
A B S T R A C T
Dedicated to the memory of Professor Emilio
Ghisalberti.
Both the traditional Chinese medicinal fungus, Antrodia camphorata, and its secondary metabolite, 4,7-di-
methoxy-5-methylbenzo[d][1,3]dioxole, have been reported to possess promising anticancer activity. In this
work the natural product and analogues bearing more polar substituents were synthesised and assessed for
antiproliferative activity in the NCI-60 screen. Although each compound inhibited the growth of some cell lines
at 10 μM, none had sufficient activity to warrant further investigation.
Keywords:
Antrodia camphorata
4,7-dimethoxy-5-methylbenzo[d][1,3]dioxole
anticancer activity
NCI-60 screen
1. Introduction
2. Results and discussion
4,7-Dimethoxy-5-methylbenzo[d][1,3]dioxole (1) (Fig. 1) is a con-
stituent of the fruiting body of the fungus Antrodia camphorata[1] (also
known as Antrodia cinnamomea, Taiwanofungus camphoratus, niu-chang-
chih, and jang-jy), which grows only on the stout camphor tree, Cinna-
momum kanehirae, a native of Taiwan. A. camphorata is a rare and
highly valued traditional Chinese medicine used for the treatment of
food and drug intoxication, diarrhoea, abdominal pain, hypertension,
itchy skin, liver diseases, and cancer [2–13]. As such, much interest has
been shown in its secondary metabolites [14–41].
The benzodioxole 1 has been shown to possess anti-inflammatory
activity, suppressing the fMLP1-induced generation of superoxide in
human neutrophils, with a potency matching ibuprofen [18], and
modestly inhibiting nitric oxide synthases [42], and other mediators of
inflammation [43].
We recently reported a synthesis of 1 and its biaryl dimer 5, which
takes advantage of a double Baeyer–Villiger oxidation of chrysazin di-
methyl ether (2) to rapidly assemble the 1,2,3,4-tetraoxygenated core
in dioxocin 3 (Scheme 1) [40].
Given the reported promising anticancer activity of the natural
product 1[42,46–48], we became interested in whether the precursor,
apiolaldehyde (4), shared its antiproliferative effects. In addition, the
formyl group provided a convenient handle for the generation of a
small series of analogues. In particular, we were interested in whether
antiproliferative activity would be maintained in more polar/water
soluble analogues that were expected to be more drug-like. The alcohol
9, apiolaldehyde (4) and apiolic acid (6) (Scheme 2), are also likely
metabolites of 1, and may be relevant to pharmacological activity of the
natural product in vivo.
In 2008, a patent claiming that 1 inhibits the growth of tumour cells
of breast cancer, liver cancer, and prostate cancer was filed [44,45].
Subsequently a number of studies have further investigated the antic-
ancer potential of the natural product [42,46–48]. In this work we
synthesised 1 and a small series of analogues and screened their anti-
proliferative activity against 60 human tumour cell lines.
The synthesis of the analogues of 1 is set out in Scheme 2. Pinnick
oxidation [49] of apiolaldehyde (4), gave apiolic acid (6). The same
conversion has previously been achieved with urea-hydrogen peroxide
[50]. The acid 6 was converted to the primary amide 7, by treatment of
the corresponding acid chloride with aqueous ammonia. Synthesis of
the nitrile 8 was achieved by direct oxidative amination [51] of apio-
laldehyde (4); this transformation has also been achieved using am-
monia and iodine [52]. Finally, the reduction of 4 with sodium bor-
ohydride gave primary alcohol 9[53].
DOI of original article: http://dx.doi.org/10.1016/j.fitote.2017.08.019
☆
A publisher's error resulted in this article appearing in the wrong issue. The article is reprinted here for the reader's convenience and for the continuity of the special issue. For
citation purposes, please use the original publication details; Fitoterapia,123, pp. 9-12. **DOI of original item: https://doi.org/10.1016/j.fitote.2017.08.019
⁎
Corresponding author.
E-mail address: matthew.piggott@uwa.edu.au (M.J. Piggott).
fMLP = N-formylmethionyl-leucyl-phenylalanine.
1
https://doi.org/10.1016/j.fitote.2018.04.001
Received 20 July 2017; Received in revised form 24 August 2017; Accepted 26 August 2017
0367-326X/©2018PublishedbyElsevierB.V.
Pleasecitethisarticleas:NoAuthor,Fitoterapia(2018),https://doi.org/10.1016/j.fitote.2018.04.001

S.Y. Yeung, M.J. Piggott
Fitoterapiaxxx(xxxx)xxx–xxx
effect of Antrodia camphorata, or that a metabolite of the natural pro-
duct is responsible for its in vivo activity anticancer activity, cannot be
ruled out at this stage.
O
O
O
4. Experimental
4.1. General details
4,7-Dimethoxybenzo[d][1,3]dioxole-5-carbaldehyde
(apiolalde-
O
hyde) (4) and 4,7-dimethoxy-5-methylbenzo[d][1,3]dioxole (1) were
synthesised as described previously [40]. Solvents were distilled before
use. ‘Petrol’ refers to the hydrocarbon fraction distilling from 64 to
67 °C; DCM = dichloromethane. Anhydrous THF (tetrahydrofuran) was
obtained from a Pure Solv 5-Mid Solvent Purification System (In-
novative Technology Inc.). All other reagents and materials were pur-
chased from commercial suppliers and used as received.
1
Fig. 1. Structure of the bioactive natural product 4,7-dimethoxy-5-methylbenzo
[d][1,3]dioxole.
The natural product 1 and analogues 4, 6–9 were assessed for an-
tiproliferative activity in the US National Cancer Institute's Human
Tumour Cell Lines Screen (NCI-60) [54] at 10 μM. Selected results are
presented in Table 1 (see Supporting information for the complete da-
taset).
Reaction progress was monitored by analytical thin layer chroma-
tography (TLC) using Merck aluminium-backed TLCF254 plates. Spots
were visualised with a UV lamp (254 nm). Organic extracts were dried
over anhydrous MgSO4. Solvents were evaporated under reduced
pressure at approximately 45 °C, and then traces of solvent were re-
moved under a flow of nitrogen.
Unfortunately, neither the natural product nor any of the analogues
showed sufficient activity against any of the 60 cell lines to warrant IC50
determinations, and any interpretation of structure–activity relation-
ships based on testing at a single concentration is therefore tentative.
Surprisingly, neither the natural product 1, which has been reported to
“profoundly decreased the growth of COLO-205 human colon cancer
cell tumor xenografts in an athymic nude mouse model” [48], nor any
of the analogues inhibited the growth of this cell line at 10 μM. The
natural product 1 was most active against SR and UO-31 cell lines,
which derive from leukemia and a renal tumour, respectively. Indeed
all analogues modestly inhibited the growth of these cell lines, with the
exception of the nitrile 8, which actually slightly promoted the growth
of UO-31 cells. In contrast, the nitrile 8 most potently inhibited the
growth of HL-60(TB) (leukemia) and HOP-92 (non-small cell lung
cancer) cell lines.
1H and 13C NMR spectra were obtained using a Varian Gemini-400
(400 MHz, 1H, 100 MHz, 13C), spectrometer. Deuterochloroform
(CDCl3) was used as the solvent, with residual CHCl3 (1H,
δ = 7.26 ppm) or CDCl3 (
13C, δ = 77.16 ppm) being used for calibra-
tion.
The infrared spectrum was recorded on a neat sample using a
Perkin-Elmer Spectrum One FT-IR spectrometer with Attenuated Total
Reflectance (ATR). Melting points were determined on a Reichert hot
stage melting point apparatus.
4.2. Synthesis
4.2.1. 4,7-Dimethoxybenzo[d][1,3]dioxole-5-carboxylic acid (apiolic
acid) (6)
Sulfamic acid (0.39 g, 1.0 mmol) was added to a stirred solution of
aldehyde 4 (0.10 g, 1.0 mmol) in acetone/water (3:1) (5 mL), followed
by sodium chlorite (0.14 g, 1.5 mmol). After 2 h, the reaction mixture
was diluted with DCM (100 mL) and the organic phase was washed
with brine (3 × 100 mL). The aqueous phase was extracted with DCM
(50 mL) and the combined organic phase was dried and evaporated to
give the carboxylic acid 6 as yellow solid (0.19 g, 83%), which crys-
tallized from DCM as off-white needles, mp = 176–178 °C [lit
[55]. = 176 °C]. Rf = 0.2 (1:1 petrol/EtOAc); 1H NMR (400 MHz,
CDCl3): δ 7.36 (s, 1H, ArH), 6.10 (s, 2H, OCH2O), 4.14 (s, 3H, OCH3),
3.88 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3): 165.3 (CHO), 141.7
3. Conclusion
The benzodioxole natural product 1, which has been reported to
have anticancer potential, and a small series of more polar analogues,
were synthesised and assessed for antiproliferative activity in the NCI-
60 screen. None of the compounds showed any significant activity at
10 μM. Hence, although 1 should not necessarily be abandoned as in-
spiration for the discovery of anticancer agents, substantial improve-
ments in potency of analogues will be required to identify a genuine
lead. The possibility that 1 contributes synergistically to an anticancer
O
O
H2 , Pd/C
O
O
O
O
O
O
O
O
O
O
O
O
CHO
AcOH
•
NaBO3 4H2O
O
O
O
1
TFA
O
O
2
3
4
O
O
O
O
O
O
O
5
Scheme 1. Previous synthesis of natural products 1 and 5[40].
2

S.Y. Yeung, M.J. Piggott
Fitoterapiaxxx(xxxx)xxx–xxx
O
O
O
O
O
O
O
NaBH4
MeOH
O
O
NaOCl2, H3NSO3
COMe2, H2O
O
O
OH
H
OH
O
O
O
9
83%
4
83%
6
Cu, NH4 Cl
O2, pyridine
1. SOCl2
2. NH3/H2O
98%
73%
O
O
O
CN
O
O
O
O
NH2
O
O
8
7
Scheme 2. Synthesis of analogues of the natural product 1.
Table 1
Percentage growth inhibitiona of selected cell lines caused by 1 and analogues at 10 μM.
#
R
Cell line
HL-60(TB)
MOLT-4
SR
HOP-92
NCI-H522
COLO 205
UO-31
MDA-MB-231/ATCC
1
4
6
7
8
9
CH3
CHO
CO2H
CONH2
CN
2
–9
–14
–1
20
5
–6
–3
5
0
24
20
20
14
11
9
n.t.b
n.t.
n.t.
1
27
n.t.
–3
11
20
0
10
15
1
–6
–4
–2
–11
–3
16
21
20
6
–11
21
–2
–11
–5
14
–17
–16
CH2OH
–12
21
a
b
Negative values indicate increases growth relative to the control.
n.t. = not tested.
(ArO), 139.9 (ArO), 138.1 (ArO), 137.8 (ArO), 113.9 (Ar), 111.4 (ArH),
103.2 (OCH2O), 61.5 (OCH3), 56.8 (OCH3).
NH), 7.45 (s, 1H, ArH), 6.07 (s, 2H, OCH2O), 5.68 (s, 1H, NH), 4.03 (s,
3H, OCH3), 3.91 (s, 3H, OCH3); 13C NMR (100 MHz, CDCl3): δ = 166.6
(CONH2), 140.1 (ArO), 139.6 (ArO), 138.8 (ArO), 137.6 (ArO), 117.8
(Ar), 110.2 (ArH), 102.7 (OCH2O), 60.8 (OCH3), 56.7 (OCH3). NMR
data have not previously been reported for this compound.
4.2.2. 4,7-Dimethoxybenzo[d][1,3]dioxole-5-carboxamide (apiolamide)
(7)
A mixture of thionyl chloride (0.5 mL, 5 mmol) and carboxylic acid
6 (0.22 g, 0.97 mmol) was stirred under reflux with moisture guard
(CaCl2) for 3 h. The excess thionyl chloride was evaporated and the
residue was dissolved in anhydrous THF (1 mL) and cooled to 0 °C, then
treated dropwise with conc. ammonia (0.4 mL). After stirring over-
night, the reaction mixture was diluted with DCM (100 mL) and the
solution was washed with water (2 × 100 mL), NaHCO3 (2 × 100 mL)
and brine (100 mL), dried and evaporated to give the amide 7 an off-
white solid (0.16 g, 73%), which crystallized from EtOAc/DCM as
yellow prisms, mp = 184–185 °C [lit [56]. (i-PrOH) = 202–203 °C].
Rf = 0.1 (1:1 petrol/EtOAc); 1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H,
4.2.3. 4,7-Dimethoxybenzo[d][1,3]dioxole-5-carbonitrile (8)
A stirred mixture of aldehyde 4 (0.21 g, 1.0 mmol), copper powder
(95 mg, 1.5 mmol), NH4Cl (0.11 g, 2.0 mmol) and pyridine (5 mL) was
heated at 100 °C under a balloon of O2 for 24 h. The reaction mixture
was allowed to cool, then diluted with 1 M HCl (100 mL) and extracted
with Et2O (5 × 50 mL). The extract was washed with brine (100 mL),
dried and evaporated to give the nitrile 8 as yellow solid (0.20 g, 98%),
which crystallized from petrol/DCM as yellow needles,
mp = 134–137 °C [lit [52]. = 134–135 °C]. Rf = 0.6 (1:1 petrol/
EtOAc); IR (ATR) cm−1: 2221 (CN); 1H NMR (400 MHz, CDCl3): δ 6.70
3

S.Y. Yeung, M.J. Piggott
Fitoterapiaxxx(xxxx)xxx–xxx
(s, 1H, ArH), 6.10 (s, 2H, OCH2O), 4.04 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3); 13C NMR (100 MHz, CDCl3): δ 144.7 (ArO), 141.2 (ArO), 139.3
(ArO), 137.8 (ArO), 116.5 (Ar), 112.3 (ArH), 102.8 (OCH2O), 97.4
(CN), 60.6 (OCH3), 57.1 (OCH3). The 1H NMR spectrum matched the
reported data [52].
effect of Antrodia camphorata extract on human lung cancer A549 cell, Proteomics 6
(2006) 826–835.
[8] H.-L. Yang, C.-S. Chen, W.-H. Chang, F.-J. Lu, Y.-C. Lai, C.-C. Chen, et al., Growth
inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia
camphorata, Cancer Lett. 231 (2006) 215–227.
[9] Y.-C. Hseu, S.-C. Chen, P.-C. Tsai, C.-S. Chen, F.-J. Lu, N.-W. Chang, et al., Inhibition
of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast
cancer cells by Antrodia camphorata, Food Chem. Toxicol. 45 (2007) 1107–1115.
[10] Y.-C. Hseu, S.-C. Chen, H.-C. Chen, J.-W. Liao, H.-L. Yang, Antrodia camphorata
inhibits proliferation of human breast cancer cells in vitro and in vivo, Food Chem.
Toxicol. 46 (2008) 2680–2688.
[11] M.-C. Lu, Y.-C. Du, J.-J. Chuu, S.-L. Hwang, P.-C. Hsieh, C.-S. Hung, et al., Active
extracts of wild fruiting bodies of Antrodia camphorata (EEAC) induce leukemia HL
60 cells apoptosis partially through histone hypoacetylation and synergistically
promote anticancer effect of trichostatin A, Arch. Toxicol. 83 (2009) 121–129.
[12] C.-H. Huang, Y.-Y. Chang, C.-W. Liu, W.-Y. Kang, Y.-L. Lin, H.-C. Chang, et al.,
Fruiting body of Niuchangchih (Antrodia camphorata) protects livers against chronic
alcohol consumption damage, J. Agric. Food Chem. 58 (2010) 3859–3866.
[13] G.-J. Huang, J.-S. Deng, C.-C. Chen, C.-J. Huang, P.-J. Sung, S.-S. Huang, et al.,
Methanol extract of Antrodia camphorata protects against lipopolysaccharide-in-
duced acute lung injury by suppressing NF-κB and MAPK pathways in mice, J.
Agric. Food Chem. 62 (2014) 5321–5329.
4.2.4. (4,7-Dimethoxybenzo[d][1,3]dioxol-5-yl)methanol (9) [53]
Sodium borohydride (45 mg, 1.2 mmol) was added portion-wise to
a stirred solution of aldehyde 4 (210 mg, 1.00 mmol) in MeOH (2.5 mL)
at 0 °C. After 5 min the reaction was quenched with water (10 mL) and
extracted with EtOAc (3 × 5 mL). The organic extract was washed with
water (5 mL), brine (5 mL), dried and evaporated to give alcohol 9 as a
colourless solid (176 mg, 83%), which crystallized from petrol/DCM as
colourless needles, mp = 86–87 °C [lit [57]. = 85 °C]; Rf = 0.35 (1:1
petrol/EtOAc); 1H NMR (400 MHz, CDCl3): δ 6.48 (s, 1H, ArH) 5.95 (s,
2H, OCH2O), 4.56 (s, 2H, CH2OH) 3.94 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 2.28 (s, 1H, OH); 13C NMR (100 MHz, CDCl3): δ = 138.8 (ArO),
138.0 (ArO), 136.5 (ArO), 136.2 (ArO), 126.1 (Ar), 107.7 (ArH), 101.7
(OCH2O), 61.6 (CH2OH), 60.1 (OCH3), 56.0 (OCH3).
[14] I.C. Yen, C.-W. Yao, M.-T. Kuo, C.-L. Chao, C.-Y. Pai, W.-L. Chang, Anti-cancer
agents derived from solid-state fermented Antrodia camphorata mycelium,
Fitoterapia 102 (2015) 115–119.
[15] N. Nakamura, A. Hirakawa, J.-J. Gao, H. Kakuda, M. Shiro, Y. Komatsu, et al., Five
new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata
and their cytotoxic effects on LLC tumor cell line, J. Nat. Prod. 67 (2004) 46–48.
[16] Y.-C. Shen, Y.-H. Wang, Y.-C. Chou, C.-F. Chen, L.-C. Lin, T.-T. Chang, et al.,
Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the
fruiting bodies of Antrodia camphorata, Planta Med. 70 (2004) 310–314.
[17] C.-C. Chen, Y.-J. Shiao, R.-D. Lin, Y.-Y. Shao, M.-N. Lai, C.-C. Lin, et al.,
Neuroprotective diterpenes from the fruiting body of Antrodia camphorata, J. Nat.
Prod. 69 (2006) 689–691.
4.3. NCI-60 screen
Details of the NCI-60 one-dose screening methodology can be found at
https://dtp.cancer.gov/discovery_development/nci-60/methodology.htm.
NSC numbers and results against the entire 60 cell lines are provided in the
Supplementary information.
[18] J.-J. Chen, W.-J. Lin, C.-H. Liao, P.-C. Shieh, Anti-inflammatory benzenoids from
Antrodia camphorata, J. Nat. Prod. 70 (2007) 989–992.
Conflicts of interest
None.
[19] T.-H. Lee, C.-K. Lee, W.-L. Tsou, S.-Y. Liu, M.-T. Kuo, W.-C. Wen, A new cytotoxic
agent from solid-state fermented mycelium of Antrodia camphorata, Planta Med. 73
(2007) 1412–1415.
[20] S.G. Stewart, M.E. Polomska, R.W. Lim, A concise synthesis of maleic anhydride and
maleimide natural products found in Antrodia camphorata, Tetrahedron Lett. 48
(2007) 2241–2244.
Acknowledgements
[21] S.-C. Chien, M.-L. Chen, H.-T. Kuo, Y.-C. Tsai, B.-F. Lin, Y.-H. Kuo, Anti-in-
flammatory activities of new succinic and maleic derivatives from the fruiting body
of Antrodia camphorata, J. Agric. Food Chem. 56 (2008) 7017–7022.
[22] S.-S. Yang, G.-J. Wang, S.-Y. Wang, Y.-Y. Lin, Y.-H. Kuo, T.-H. Lee, New constituents
with iNOS inhibitory activity from mycelium of Antrodia camphorata, Planta Med.
75 (2009) 512–516.
[23] Y.-C. Hsieh, Y.K. Rao, J. Whang-Peng, C.-Y.F. Huang, S.-K. Shyue, S.-L. Hsu, et al.,
Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in
hepatocellular carcinoma cells involves enhancing oxidative stress coincident with
activation of intrinsic and extrinsic apoptotic pathway, J. Agric. Food Chem. 59
(2011) 10943–10954.
[24] C.-L. Lee, C.-H. Huang, H.-C. Wang, D.-W. Chuang, M.-J. Wu, S.-Y. Wang, et al.,
First total synthesis of antrocamphin A and its analogs as anti-inflammatory and
anti-platelet aggregation agents, Org. Biomol. Chem. 9 (2011) 70–73.
[25] K.A. Punch, E.L. Ghisalberti, M.J. Piggott, Is 2,3,4,5-tetramethoxybenzoyl chloride
a natural product? J. Nat. Prod. 74 (2011) 1348–1350.
[26] Y.-R. Liao, P.-C. Kuo, S.-C. Huang, J.-W. Liang, T.-S. Wu, An efficient total synthesis
of benzocamphorin H and its anti-inflammatory activity, Tetrahedron Lett. 53
(2012) 6202–6204.
We gratefully acknowledge a summer vacation scholarship for S–Y.
Y from the Cancer Council Western Australia. We thank the National
Cancer Institute (NCI) Developmental Therapeutics Program (https://
dtp.cancer.gov) for the screening data. The graphical abstract image is
used with permission from Mushroom Nutrition, www.
mushroomnutrition.com. MJP is forever grateful to Professor Emil
Ghisalberti for importing his passion for natural products research, and
for placing his students' interests above his own.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.fitote.2017.08.019.
[27] J.-L. Rios, I. Andujar, M.-C. Recio, R.-M. Giner, Lanostanoids from fungi: a group of
potential anticancer compounds, J. Nat. Prod. 75 (2012) 2016–2044.
[28] J.-S. Deng, S.-S. Huang, T.-H. Lin, M.-M. Lee, C.-C. Kuo, P.-J. Sung, et al., Analgesic
and anti-inflammatory bioactivities of eburicoic acid and dehydroeburicoic acid
isolated from Antrodia camphorata on the inflammatory mediator expression in
mice, J. Agric. Food Chem. 61 (2013) 5064–5071.
[29] C.-C. Liaw, Y.-C. Chen, G.-J. Huang, Y.-C. Tsai, S.-C. Chien, J.-H. Wu, et al., Anti-
inflammatory lanostanoids and lactone derivatives from Antrodia camphorata, J.
Nat. Prod. 76 (2013) 489–494.
References
[1] H.-C. Chiang, D.-P. Wu, I.W. Cherng, C.-H. Ueng, A sesquiterpene lactone, phenyl
and biphenyl compounds from Antrodia cinnamomea, Phytochemistry 39 (1995)
613–616.
[2] M. Geethangili, Y.-M. Tzeng, Review of pharmacological effects of Antrodia cam-
phorata and its bioactive compounds, Evid. Based Complement. Alternat. Med. 2011
(2011) 212641.
[30] M. Buccini, K.A. Punch, B. Kaskow, G.R. Flematti, B.W. Skelton, L.J. Abraham,
et al., Ethynylbenzenoid metabolites of Antrodia camphorata: synthesis and inhibi-
tion of TNF expression, Org. Biomol. Chem. 12 (2014) 1100–1113.
[31] Y. Huang, X. Lin, X. Qiao, S. Ji, K. Liu, C.-T. Yeh, et al., Antcamphins A-L, ergos-
tanoids from Antrodia camphorata, J. Nat. Prod. 77 (2014) 118–124.
[32] H.-M. Lien, C.-J. Tseng, C.-L. Huang, Y.-T. Lin, C.-C. Chen, Y.-Y. Lai, Antimicrobial
activity of Antrodia camphorata extracts against oral bacteria, PLoS One 9 (2014)
(e105286/105281-e105286/105287, 105287pp.).
[3] G. Hsiao, M.-Y. Shen, K.-H. Lin, M.-H. Lan, L.-Y. Wu, D.-S. Chou, et al.,
Antioxidative and hepatoprotective effects of Antrodia camphorata extract, J. Agric.
Food Chem. 51 (2003) 3302–3308.
[4] Y.-L. Hsu, Y.-C. Kuo, P.-L. Kuo, L.-T. Ng, Y.-H. Kuo, C.-C. Lin, Apoptotic effects of
extract from Antrodia camphorata fruiting bodies in human hepatocellular carci-
noma cell lines, Cancer Lett. 221 (2005) 77–89.
[5] T.-Y. Song, S.-L. Hsu, C.-T. Yeh, G.-C. Yen, Mycelia from Antrodia camphorata in
submerged culture induce apoptosis of human hepatoma HepG2 cells possibly
through regulation of Fas pathway, J. Agric. Food Chem. 53 (2005) 5559–5564.
[6] C.-C. Peng, K.-C. Chen, R.Y. Peng, C.-H. Su, H.M. Hsieh-Li, Human urinary bladder
cancer T24 cells are susceptible to the Antrodia camphorata extracts, Cancer Lett.
243 (2006) 109–119.
[33] R.S. Sulake, Y.-F. Jiang, H.-H. Lin, C. Chen, Total synthesis of ( )-antroquinonol
D, J. Org. Chem. 79 (2014) 10820–10828.
[34] S.-C. Wang, T.-H. Lee, C.-H. Hsu, Y.-J. Chang, M.-S. Chang, Y.-C. Wang, et al.,
Antroquinonol D, isolated from Antrodia camphorate, with DNA demethylation and
anticancer potential, J. Agric. Food Chem. 62 (2014) 5625–5635.
[35] W.-H. Chang, M.C. Chen, I.H. Cheng, Antroquinonol lowers brain amyloid-β levels
[7] H. Wu, C.-L. Pan, Y.-C. Yao, S.-S. Chang, S.-L. Li, T.-F. Wu, Proteomic analysis of the
4

S.Y. Yeung, M.J. Piggott
Fitoterapiaxxx(xxxx)xxx–xxx
and improves spatial learning and memory in a transgenic mouse model of
Evid. Based Complement. Alternat. Med. 2011 (2011) 450529.
Alzheimer's disease, Sci. Rep. 5 (2015) 15067.
[47] H.-M. Lien, H.-W. Lin, Y.-J. Wang, L.-C. Chen, D.-Y. Yang, Y.-Y. Lai, et al., Inhibition
of anchorage-independent proliferation and G0/G1 cell-cycle regulation in human
colorectal carcinoma cells by 4,7-dimethoxy-5-methyl-l,3-benzodioxole isolated
from the fruiting body of Antrodia camphorate, Evid. Based Complement. Alternat.
Med. 2011 (2011) 984027.
[36] W.-H. Chang, H. Cheng Irene, C. Chen Miles, H. Cheng Irene, H. Cheng Irene,
Antroquinonol lowers brain amyloid-β levels and improves spatial learning and
memory in a transgenic mouse model of Alzheimer's disease, Sci. Rep. 5 (2015)
15067.
[37] Y.-H. Kuo, C.-H. Lin, C.-C. Shih, Antidiabetic and antihyperlipidemic properties of a
triterpenoid compound, dehydroeburicoic acid, from Antrodia camphorata in vitro
and in streptozotocin-induced mice, J. Agric. Food Chem. 63 (2015) 10140–10151.
[38] Y.-H. Kuo, C.-H. Lin, C.-C. Shih, Ergostatrien-3β-ol from Antrodia camphorata in-
hibits diabetes and hyperlipidemia in high-fat-diet treated mice via regulation of
hepatic related genes, glucose transporter 4, and AMP-activated protein kinase
phosphorylation, J. Agric. Food Chem. 63 (2015) 2479–2489.
[39] Y.-H. Kuo, T.-Y. Lin, Y.-J. You, K.-C. Wen, P.-J. Sung, H.-M. Chiang,
Antiinflammatory and antiphotodamaging effects of ergostatrien-3β-ol, isolated
from Antrodia camphorata, on hairless mouse skin, Molecules 21 (2016) 1213/
1211–1213/1214.
[40] H.L. Newson, D.A. Wild, S.Y. Yeung, B.W. Skelton, G.R. Flematti, J.E. Allan, et al.,
Access to 1,2,3,4-tetraoxygenated benzenes via a double Baeyer–Villiger reaction of
quinizarin dimethyl ether: application to the synthesis of bioactive natural products
from Antrodia camphorata, J. Org. Chem. 81 (2016) 3127–3135.
[41] H.-C. Lin, M.-H. Lin, J.-H. Liao, T.-H. Wu, T.-H. Lee, F.-L. Mi, et al., Antroquinonol, a
ubiquinone derivative from the mushroom Antrodia camphorata, inhibits colon
cancer stem cell-like properties: insights into the molecular mechanism and in-
hibitory targets, J. Agric. Food Chem. 65 (2017) 51–59.
[48] S.-H. Tu, C.-H. Wu, L.-C. Chen, C.-S. Huang, H.-W. Chang, C.-H. Chang, et al., In vivo
antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the
fruiting body of Antrodia camphorata through activation of the p53-mediated p27/
Kip1 signaling pathway, J. Agric. Food Chem. 60 (2012) 3612–3618.
[49] P.T. Gunning, Positive ion pair cooperativity exhibited for the binding of phosphate
under physiological conditions, Org. Biomol. Chem. 3 (2005) 3877–3879.
[50] V.V. Semenov, A.S. Kiselyov, I.Y. Titov, I.K. Sagamanova, N.N. Ikizalp,
N.B. Chernysheva, et al., Synthesis of antimitotic polyalkoxyphenyl derivatives of
combretastatin using plant allylpolyalkoxybenzenes, J. Nat. Prod. 73 (2010)
1796–1802.
[51] P. Capdevielle, A. Lavigne, M. Maumy, Simple and efficient copper-catalyzed one-
pot conversion of aldehydes into nitriles, Synthesis 1989 (1989) 451–452.
[52] L.D. Konyushkin, T.I. Godovikova, S.K. Vorontsova, D.V. Tsyganov, I.B. Karmanova,
M.M. Raihstat, et al., Polyalkoxybenzenes from plant raw materials 4. Parsley and
dill seed extracts in the synthesis of polyalkoxy-3,5-diaryl-1,2,4-oxadiazoles with
antiproliferative activity, Russ. Chem. Bull. 59 (2010) 2268–2275.
[53] D.V. Tsyganov, L.D. Konyushkin, I.B. Karmanova, S.I. Firgang, Y.A. Strelenko,
M.N. Semenova, et al., cis-Restricted 3-aminopyrazole analogues of com-
bretastatins: synthesis from plant polyalkoxybenzenes and biological evaluation in
the cytotoxicity and phenotypic sea urchin embryo assays, J. Nat. Prod. 76 (2013)
1485–1491.
[42] L.-S. Shi, C.-H. Chao, D.-Y. Shen, H.-H. Chan, C.-H. Chen, Y.-R. Liao, et al.,
Biologically active constituents from the fruiting body of Taiwanofungus camphor-
atus, Bioorg. Med. Chem. 19 (2011) 677–683.
[43] P.-H. Shie, S.-Y. Wang, H.-L. Lay, G.-J. Huang, 4,7-Dimethoxy-5-methyl-1,3-ben-
zodioxole from Antrodia camphorata inhibits LPS-induced inflammation via sup-
pression of NF-κB and induction HO-1 in RAW264.7 cells, Int. Immunopharmacol.
31 (2016) 186–194.
[54] NIH National Cancer Institute, Division of Cancer Treatemtn & Diagnosis (Accessed
18/07/2017),, 2017. https://dtp.cancer.gov/discovery_development/nci-60/, .
[55] F. Dallacker, G. Reichrath, G. Schnackers, Spasm activity caused by benzo- and
naphtho-1.3-dioxolecarboxylic acids in mice, Z. Naturforsch., C: Biosci. 35C (1980)
49–56.
[44] US Pat., 2007-749659, 20080103195, 2008.
[45] CN Pat., 2007-10001559, 101214238, 2008.
[46] H.-M. Lien, P.-T. Kuo, C.-L. Huang, J.-Y. Kao, H. Lin, D.-Y. Yang, et al., Study of the
anti-proliferative activity of 5-substituted 4,7-dimethoxy-1,3-benzodioxole deriva-
tives of SY-1 from Antrodia camphorata on human COLO 205 colon cancer cells,
[56] F. Dallacker, F. Gohlke, Derivatives of methylenedioxybenzene. VIII. Synthesis of
apiolic and 2-methoxyapiolic acids, Justus Liebigs Ann. Chem. 644 (1961) 30–36.
[57] F. Dallacker, W. Imoehl, M. Pauling-Walther, Derivatives of methylenediox-
ybenzene. XI. Preparation of hydroxymethyl derivatives of methylenediox-
ybenzene, Justus Liebigs Ann. Chem. 681 (1965) 111–117.
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