Synthesis of the proposed structure of damaurone D and evaluation of its anti-inflammatory activity

Article abstract of DOI:10.1248/cpb.c15-00528

Concise and efficient synthesis of the proposed structure of damaurone D is accomplished in five steps without protection-deprotection operations. The key feature of our synthesis includes a versatile aldol reaction of the benzofuranone, provided by selec

Full text of DOI:10.1248/cpb.c15-00528

Vol. 63, No. 11
Chem. Pharm. Bull. 63, 907–912 (2015)
907
Regular Article
Synthesis of the Proposed Structure of Damaurone D and Evaluation of
Its Anti-inflammatory Activity
,a
Young Taek Han,* Zheng Wang,b and Eun Ju Baeb
b
a College of Pharmacy, Dankook University; Cheonan 330–141, Korea: and College of Pharmacy, Woosuk
University; Wanju 565–701, Korea.
Received July 2, 2015; accepted August 11, 2015; advance publication released online August 20, 2015
Concise and efficient synthesis of the proposed structure of damaurone D is accomplished in five steps
without protection-deprotection operations. The key feature of our synthesis includes a versatile aldol reac-
tion of the benzofuranone, provided by selective α-halogenation and intramolecular O-alkylation. However,
the H- and C-NMR spectral data of the synthesized damaurone D did not agree with previous reports. The
structure of the synthesized damaurone D was confirmed using combined two dimensional (2D)-NMR analy-
sis, including heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity
(HMBC), and nuclear Overhauser effect spectroscopy (NOESY). The synthesized damaurone D was found
to exhibit potent anti-inflammatory activity in murine macrophage RAW264.7 cells, which was demonstrated
by the findings that damaurone D treatment in cells resulted in the inhibition of lipopolysaccharide (LPS)-
stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and nitrite pro-
duction.
Key words damaurone D; synthesis; anti-inflammatory activity; aurone; selective α-bromination
Aurones [2-benzylidenebenzofuran-3(2H)-ones], flavonoid aurone moiety may be a useful novel scaffold for further me-
constituents of the yellow pigmentation in flowers and fruits,1) dicinal chemistry research such as the construction of natural
exhibit a strong and broad variety of biological efficacies product-like libraries.
such as fungicidal, antiviral, anti-tyrosinase and antioxidant
To establish a synthetic procedure for damaurone D (1), that
activities.2) In this regard, these aurones and their derivatives could be used for further derivatization, we planned a versa-
have been considered as attractive targets in terms of both tile aldol condensation between benzofuranone 2 and 4-hy-
synthetic3) and medicinal chemistry.4,5) Recently, damaurone droxybenzaldehyde, a general synthetic approach for Z-aurone
D (1), an isoprenylated aurone, was isolated by Lei et al. derivatives.12,13) Benzofuranone 2 was expected to be obtained
from Rosa damascena, a plant that belongs to genus Rosa.6) by α-halogenation, followed by intramolecular O-alkylation
The genus Rosa including Rosa damascena has not only been of 2-hydroxyacetophenone 3, which could be prepared from
used for gardening and cosmetics, but also as a traditional commercially available 2,4-dihydroxyacetophenone 4 by regi-
medication to treat depression, inflammation, and infectious oselective chromene annulation and subsequent hydrogenation.
diseases.7) Based on these interesting biological activities, Alternatively, benzofuranone 2 was expected to be prepared
numerous research including identification of novel drug by chromene annulation followed by hydrogenation of com-
candidates, as well as its applications, have been performed mercially available hydroxycoumaranone 5 (Fig. 1).
using the extracts and single chemical constituents of Rosa
Natural compounds that have an enone structure between
damascena.8–10) Considering not only the therapeutic proper- two aromatic rings such as chalcones and aurones are gener-
ties of aurones and Rosa damascena as mentioned above, but ally considered as Michael acceptors which can covalently
also the existence of a chromane moiety, one of the privileged bind biomolecules, and exhibit interesting pharmacological
structures in medicinal chemistry,11) damaurone D is strongly activities.14) For instance, butein, a polyphenolic chalcone, and
expected to have therapeutically useful biological activities. sulfuretin, one of the aurones isolated from Rhus verniciflua,
Furthermore, it is believed that its unique dihydropyrano- have been shown to possess an anti-inflammatory function in
Fig. 1. Proposed Structure of Damaurone D (1) and Retrosynthetic Analysis
*To whom correspondence should be addressed. e-mail: hanyt@dankook.ac.kr
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 11 (2015)
vitro and in vivo.15–17) Given that damaurone D (1) has similar also anticipated that a substrate possessing more nucleophilic
enone structure to the natural anti-inflammatory compounds, carbonyl α-carbon than aromatic rings would undergo an
herein, we examine whether damaurone D (1) exerts anti- α-bromination reaction selectively. Based on this idea, we
inflammatory action in macrophages.
performed a two-step α-bromination via trimethylsilyl (TMS)
enol ether 10, equivalent to a highly nucleophilic enolate,24)
and observed the α-bromination product 7 as expected (entry
Results and Discussion
As shown in Chart 1, our synthesis commenced with prepa- 5). Benzofuranone 2 was readily obtained in moderate yield
ration of benzofuranone 2, a precursor to the versatile aldol from crude bromoacetophenone 7, with a trace amount of 9
reaction. Pyridine-catalyzed condensation18) under microwave (<3%), by an intramolecular O-alkylation reaction. Finally,
irradiation between 2,4-dihydroxyacetophenone 4 and 3-meth- we successfully synthesized the proposed structure of dam-
yl-2-butenal regioselectively gave the known chromene 6,19) aurone D 1 by aldol condensation between benzofuranone 2
which was subjected to hydrogenation using palladium on car- and 4-hydroxybenzaldehyde using piperazine as a catalyst.
bon as a catalyst to afford chromane 3. This two-step proce- The structure of synthesized 1 was assigned and confirmed by
dure to obtain the chromane structure was more regioselective two dimensional (2D)-NMR analysis including heteronuclear
than previously described one-step reaction using isoprene.20) multiple quantum coherence (HMQC), heteronuclear multiple
In addition, microwave irradiation enhanced the reaction bond connectivity (HMBC), and nuclear Overhauser effect
rate with a slightly improved chemical yield than the reflux spectroscopy (NOESY). Selected HMBC correlations and our
conditions described in a previous study.19) However, inter- assignments are shown in Fig. 2A and Table 2, respectively.
estingly, chromene annulations of hydroxycoumaranone 5 to The geometry of the double bond was confirmed as Z con-
synthesize chromene 8 under microwave irradiation and reflux
conditions afforded only a mixture of aldol adducts. This may
be attributed to the difference in nucleophilicity of the car-
bonyl α-carbon between the 3-coumaranone and acetophenone
structures. To prepare the key intermediate benzofuranone 2
from 2-hydroxyacetophenone 3, as shown in Table 1, we at-
tempted α-bromination reactions using several brominating
reagents such as copper(II) bromide,21) N-bromosuccinimide
(NBS),22) and bromine.23) However, we could obtain only in-
separable regioisomeric mixtures of the undesired aromatic
bromination product 9 in all our experiments carried out in
direct α-bromination conditions (entries 1–4) that are gener-
ally used for the preparation of a benzofuran-3-one structure
from 2-hydroxyacetophenones. It was supposed that the nu-
cleophilicity of the aromatic ring of 2-hydroxyacetophenone
3 was increased enough by electron-donating substituents
to overcome the reactivity of the carbonyl α-carbon. It was
Table 1. Bromination Reactions of 2-Hydroxyacetophenone 3
Entry
Condition
CuBr2 (2eq), EtOH, reflux
Result
1
2
3
4
5
Only 9a)
CuBr2 (2eq), EtOAc/CHCl3, reflux
NBS (1.1eq), TsOH (1.5eq), CH3CN, reflux
Br2 (1.2eq), CHCl3, heating
1) LHMDS (3eq), TMSCl (4eq), THF, −78°C
2) NBS (1.1eq), −78°C to rt
7b)
a) Yields were not determined. b) Trace amount of 9 (<3%) was isolated after
intramolecular O-alkylation reaction.
Reagents and conditions: (a) 3-Methyl-2-butenal, pyridine, microwave irradiation, 140°C, 2h, 71%; (b) 10% Pd/C, H2, EtOAc, rt, 99%; (c) 1M LHMDS in THF, TMSCl,
THF, −78°C, 4h; then N-bromosuccinimide, −78°C→rt, 1h; (d) 1M NaOH, H2O, rt, 1h, 64% in 2 steps; (e) 4-Hydroxybenzaldehyde, piperazine, EtOH, reflux, overnight,
94%.
Chart 1

Vol. 63, No. 11 (2015)
Chem. Pharm. Bull.
909
With the proposed structure of damaurone D (1) in hand,
we examined its anti-inflammatory activity as a preliminary
evaluation of its biological activities. Firstly, we determined
the non-cytotoxic concentration range of 1 in RAW264.7
macrophage cells. As shown in Fig. 3A, the cell viability in
RAW264.7 cells was not affected by 1 up to a concentration of
20µM, thus, subsequent experiments were conducted at 10 and
20µM of 1. We next examined the concentration-dependent
effect on inducible nitric oxide synthase (iNOS) and cyclooxy-
genase-2 (COX-2) induction. Lipopolysaccharide (LPS) treat-
ment for 24h in RAW264.7 cells led to the marked induction
of iNOS and COX-2, however, pretreatment with 1 at 10 and
20µM for 1h significantly suppressed the expression of iNOS
and COX-2, being equipotent to butein (Fig. 3B). Consistently,
nitrite production in response to LPS treatment was signifi-
cantly inhibited by pretreatment with 1 (Fig. 3C) indicating
synthesized damaurone D (1) has potent anti-inflammatory
efficacy.
Fig. 2. Selected HMBC Correlation (A) and NOE Correlation (B) for
Synthesized Damaurone D
Table 2. 1H- and 13C-NMR Assignments of Damaurone D (1)
Reported
Synthesizedb)
Positiona)
1H-NMR
13C-NMR
1H-NMR
13C-NMR
2
—
—
146.1
181.3
121.0
113.0
164.0
115.4
160.6
113.9
112.2
21.2
—
—
147.1
182.7
123.0
114.3
161.8
106.2
165.9
114.2
112.3
16.0
3
4
7.39 (d)
6.66 (d)
—
7.66 (d)
6.73 (d)
—
5
6
7
—
—
Conclusion
8
9
—
—
In summary, concise and efficient synthesis of the proposed
structure of damaurone D was accomplished in five steps
without protection-deprotection operations. The key interme-
diate benzofuranone 2, which can be used as a versatile inter-
mediate for the synthesis of various dihydropyrano-aurones,
was provided by selective α-halogenation, and subsequent
intramolecular O-alkylation. The spectral data of the synthe-
sized damaurone D did not agree with that in previous report.
Structure of synthesized damaurone D was confirmed by
combined 2D-NMR analysis, including HSQC and HMBC.
Synthesized damaurone D was found to exhibit potent anti-
inflammatory activity in murine macrophage RAW264.7 cells.
On the basis of these results, further biological evaluation of
1 and its derivatives as potential anti-inflammatory agents is
currently making good progress, and results will be reported
—
—
10
6.62 (s)
2.58 (dd)
1.72 (dd)
—
7.17 (s)
2.67 (t)
1.70 (t)
—
11
12
33.1
31.3
13
73.9
76.6
14
1.52 (s)
1.52 (s)
—
26.3
1.27 (s)
1.27 (s)
—
26.6
15
26.3
26.6
1′
126.3
131.1
116.3
158.2
—
124.4
134.1
117.1
161.0
—
2′,6′
3′,5′
4′
7.82 (d)
6.89 (d)
—
8.07 (d)
7.33 (d)
—
HO-C(4′)
11.20 (s)
12.46 (s)
a) C-Atom numbering as indicated in Fig. 1. b) Pyridine-d5 was used as the sol-
vent.
figuration by the NOE correlation between benzylic protons in due course.
(H-11) and aromatic protons (H-2′,6′) as shown in Fig. 2B. In
addition, the Z configuration of the double bond in aurones
Experimental
also can be assigned by chemical shift of C-10 (112.3ppm) in
General Unless noted otherwise, all starting materials
a range of 105.9–112.8ppm. On the other hand, C-10 carbon and reagents were obtained commercially and were used with-
signal of the alternative E configuration would be observed out further purification. Tetrahydrofuran was distilled from
1
between 119.9 and 121.5ppm.6,25) However, H- and 13C-NMR sodium benzophenone ketyl. All solvents used for routine
data of 1 slightly differ from the reported data in spite of the product isolation and chromatography were of reagent grade
same measurement conditions (pyridine-d5 as the solvent and and glass distilled. Reaction flasks were dried at 100°C before
500MHz as measurement frequency). The proton signal of use, and air and moisture sensitive reactions were performed
H-10 (numbering as indicated in Fig. 1) was reported in the under argon. Flash column chromatography was performed
previous study as 6.62ppm (s, 1H), the most up-field shifted using silica gel 60 (230–400 mesh, Merck) with the indicated
peak among the aromatic and olefinic proton peaks. However, solvents. Thin-layer chromatography was performed using
in our study, the singlet proton peak of H-10 was observed at 0.25mm silica gel plates (Merck). Mass spectra were obtained
7.17ppm, very close to the proton peaks of pyridine, between using a VG Trio-2 GC-MS instrument, and high resolution
the peaks of H-3′, H-5′ (7.33ppm) and H-5 (6.73ppm). When mass spectra were obtained using a JEOL JMS-AX 505WA
we changed the NMR solvent to CD3OD, the singlet proton unit. Infrared spectra were recorded on a Fourier transform
1
peak of H-10 (6.78ppm) was also observed between the peaks (FT)-IR spectrometer. H and 13C spectra were recorded on
of H-3′, H-5′ (6.88ppm) and H-5 (6.62ppm). In addition, the a JEOL JNM-LA 300, Bruker Analytik ADVANCE digital
C-11 carbon signal in our study (16.0ppm) showed a differ- 400 or ADVANCE digital 500 in deuteriochloroform (CDCl3),
ence of more than 5ppm in comparison with that previously deuteriomethanol (CD3OD) or deuterated pyridine (pyridine-
reported (21.2ppm). Considering 13C-NMR signals of the d5). Chemical shifts are expressed in parts per million (ppm,
corresponding carbons of synthetic intermediates such as δ) downfield from tetramethylsilane and are referenced to the
1
chromanes 3 (16.2ppm) and 2 (15.7ppm), prepared from the deuterated solvents. H-NMR data are reported in the order;
known chromene 6, our assignment of the C-11 carbon seems chemical shift, multiplicity (s, singlet; brs, broad singlet; d,
to be more reasonable.
doublet; t, triplet; q, quartet; m, multiplet, and/or multiple res-

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Chem. Pharm. Bull.
Vol. 63, No. 11 (2015)
Fig. 3. (A) Effect of Synthesized Damaurone D (1) on Cell Viability of RAW264.7 Macrophages; (B) 1 Inhibited iNOS and COX-2 Protein Expres-
sion in RAW264.7 Cells; (C) Nitrite Production Was Measured in the Cells from (B)
(A) RAW264.7 cells were treated with 1 at 10, 20 and 40µM for 24h in the presence of serum, and subsequently subjected to an MTT assay. The results are expressed as
the mean S.E.M. (B) Cells were treated with LPS (10ng/mL) in the absence or presence of 1 or butein at 10 and 20µM for 24h. β-Tubulin was used as a loading control.
The experiments were repeated at least three times, and the relative levels of iNOS and COX-2 expression analyzed by densitometry are shown in the lower bar graph.
**p<0.01 vs. LPS-treated group.
onance, numbers of protons, and coupling constants in hertz flash column chromatography on silica gel (EtOAc–CH2Cl2–n-
(Hz). HMQC and HMBC were recorded on Bruker Avance III hexane=1:1: 15) afforded 999mg (99%) of chromane 3 as a
700MHz in deuterated pyridine.
white solid with a melting point of 68–71°C: FT-IR (thin film,
1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)ethanone neat) νmax 2976, 1627, 1586, 1370, 1270, 1118cm−1; H-NMR
(6) To a solution of 2,4-dihydroxyacetophenone 4 (2.00g, (400MHz, CDCl3) δ: 13.08 (s, 1H), 7.46 (d, 1H, J=8.8Hz),
13.14mmol) in pyridine (2mL) was added 3-methyl-2-butenal 6.31 (d, 1H, J=8.9Hz), 2.67 (t, 2H, J=6.8Hz), 2.52 (s, 3H),
(1.65mL, 17.09mmol) at ambient temperature. The reaction 1.79 (t, 2H, J=6.8Hz), 1.33 (s, 6H); 13C-NMR (75MHz,
mixture was stirred for 2h at 140°C under microwave irra- CDCl3) δ: 202.5, 162.7, 160.7, 129.5, 112.6, 109.1, 109.5, 75.7,
diation and cooled to room temperature. The reaction mixture 31.8, 26.7, 26.7, 26.0, 16.2; LR-MS (FAB) m/z 221 (M+H+);
was diluted with EtOAc and 2N HCl solution. The organic HR-MS (FAB) Calcd for C13H17O3 (M+H+): 221.1172. Found
layer was washed with water and brine, dried over MgSO4 221.1189.
1
and concentrated in vacuo. Purification of the residue via flash
7,7-Dimethyl-8,9-dihydro-2H-furo[2,3-f]chromen-3(7H)-
column chromatography on silica gel (EtOAc–n-CH2Cl2–hex- one (2) To a solution of 2′-hydroxyacetophenone 3 (400mg,
ane=1:1:20–1:1:15) afforded 2.03g (71%) of the chromene 1.81mmol) in tetrahydrofuran (THF) (20mL) was dropwised
6 as pale yellow solid with a melting point of 101 ca. 103°C: 1M lithium hexamethyldisilazide (LHMDS) solution in THF
FT-IR (thin film, neat) νmax 2976, 1644, 1624, 1578, 1487, (5.45mL, 5.45mmol) at −78°C. After 30min, trimethylsilyl
1
1365, 1273, 1113cm−1; H-NMR (400MHz, CDCl3) δ: 12.94 chloride (TMSCl) (0.92mL, 7.26mmol) was added and the
(s, 1H), 7.49 (d, 1H, J=8.2Hz), 6.69 (d, 1H, J=10.0Hz), 6.31 resulting mixture was stirred for 4h at same temperature. N-
(d, 1H, J=9.2Hz), 5.56 (d, 1H, J=10.0Hz), 2.52 (s, 3H), 1.43 Bromosuccinimide (355mg, 1.99mmol) in THF (5mL) was
(s, 6H); low resolution (LR)-MS (FAB) m/z 219 (M+H+); dropwised for 10min and the resulting mixture was stirred
high resolution (HR)-MS (FAB) Calcd for C13H15O3 (M+H+): for 30min at −78°C and 30min at ambient temperature.
219.1016. Found 219.1029.
To the reaction mixture was added 1M sodium hydroxide
1-(5-Hydroxy-2,2-dimethylchroman-6-yl)ethanone
(3) (NaOH) solution (10mL) at ambient temperature and stirred
A suspension of chromene 6 (1.00g, 4.58mmol) and 10% Pd/C for 1h. The reaction mixture was diluted with EtOAc and
(50mg) in EtOAc (50mL) was stirred under H2 atmosphere at water. The organic layer was washed with water and brine,
ambient temperature until no starting material was detected dried over MgSO4 and concentrated in vacuo. Purification
by TLC. The reaction mixture was filtered through a Celite of the residue via flash column chromatography on silica gel
pad and concentrated in vacuo. Purification of the residue via (EtOAc–n-hexane–CH2Cl2=1:1:10) afforded 253mg (64%)

Vol. 63, No. 11 (2015)
Chem. Pharm. Bull.
911
of the benzofuranone 2 as pale yellow solid with a melting U.S.A.). The aliquots of lysates were eletrophoresed in 6–10%
point of 148–150°C, and trace amount of the bromoacetophe- sodium dodecyl sulfate-polyacrylamide gels (20µg of protein/
none 9: FT-IR (thin film, neat) νmax 2974, 1974, 1705, 1599, lane). The separated proteins were transferred to nitrocellulose
1
1440, 1318cm−1; H-NMR (400MHz, CDCl3) δ: 7.35 (d, 1H, membranes (GE Healthcare). The membranes were blocked
J=8.8Hz), 6.51 (d, 1H, J=8.3Hz), 4.69 (s, 2H), 2.73 (t, 2H, with 0.4% skim milk in Tris-buffered saline containing 1%
J=6.8Hz), 1.86 (t, 2H, J=6.8Hz); 1.36 (s, 6H); 13C-NMR Tween 20 and incubated with the primary antibodies, followed
(75MHz, CDCl3) δ: 197.7, 174.1, 162.5, 122.3, 113.4, 113.0, by incubation with secondary antibodies. Immunoreactive
105.7, 76.2, 75.6, 31.4, 26.7, 26.7, 15.7; LR-MS (FAB) m/z protein was visualized by ECL chemiluminescence detection
219 (M+H+); HR-MS (FAB) Calcd for C13H15O3 (M+H+): kit (Amersham Biosciences, Buckinghamshire, U.K.). Image
219.1016. Found 219.1021.
was obtained using ChemiDoc™ XRS+ System (Bio-Rad) or
1-[7(or 8)-Bromo-5-hydroxy-2,2-dimethylchroman-6-yl]- conventional developing method using Kodak film. Primary
ethanone (9) 1H-NMR (400MHz, CDCl3, mixture of regioi- antibodies used were: iNOS from BD Biosciences (Palo Alto,
somers) δ: 7.76 and 7.58 (1H, s), 2.73–2.66 (m, 2H), 2.56–2.54 CA, U.S.A.), β-tubulin (#PA1-16947) from Thermo Scien-
(m, 3H), 1.85–1.81 (m, 2H), 1.36–1.34 (m, 6H); LR-MS (FAB) tific (Waltham, MA, U.S.A.), and COX-2 (sc-1745) from Santa
m/z 299 (M+H+).
(Z)-2-(4-Hydroxybenzylidene)-7,7-dimethyl-8,9-di-
Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
Measurement of Nitric Oxide (NO) Levels Production of
hydro-2H-furo[2,3-f]chromen-3(7H)-one (Damaurone D, NO was estimated by measuring the amount of nitrite (a stable
1) To a solution of benzofuranone 2 (100mg, 0.46mmol) metabolite of NO) using the Griess reagent. Briefly, cells
and 4-hydroxybenzaldehyde (56mg, 0.46mmol) in anhydrous were pretreated with DD for 1h before the addition of LPS.
EtOH (10mL) was added piperazine (4.3mg, 0.05mmol). After 24h, aliquots of culture supernatants were mixed with
The reaction mixture was refluxed for 24h and then diluted an equal volume of a modified Griess reagent comprising a
with EtOAc and saturated NH4Cl aqueous solution. The or- 1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1%
ganic layer was washed with water and brine, dried over N-(1-naphthyl)ethylenediamine dihydrochloride in 60% acetic
MgSO4 and concentrated in vacuo. Purification of the residue acid, at room temperature for 5min, and absorbance was mea-
via flash column chromatography on silica gel (MeOH– sured at 540nm using a spectrophotometer.
CH2Cl2=1:100–1:50) afforded 139mg (94%) of the damau-
Statistical Analyses Data are provided as mean standard
rone D (1) as yellow solid with a melting point of 276–279°C; error of the mean (S.E.M.) values. The significance of differ-
FT-IR (thin film, neat) νmax 1680, 1595, 1513, 1437, 1281, 1130, ences between treatment groups was determined by one-way
1059 cm−1; 1H-NMR (500MHz, pyridine-d5) δ: 12.46 (brs, ANOVA with the Turkey’s post-hoc test using GraphPad
1H), 8.07 (d, 2H, J=8.6Hz), 7.66 (d, 1H, J=8.5Hz), 7.33 (d, Prism v4.0 (GraphPad, San Diego, CA, U.S.A.). p<0.01 was
2H, J=8.6Hz), 7.17 (s, 1H), 6.73 (1H, d, J=8.4Hz), 2.67 (t, considered significant.
1
2H, J=6.7Hz), 1.70 (t, 2H, J=6.7Hz), 1.27 (s, 6H); H-NMR
(400MHz, CD3OD) δ: 7.81 (d, 2H, J=8.8Hz), 7.48 (d, 1H,
Acknowledgment The present research was conducted by
J=8.8Hz), 6.88 (d, 2H, J=8.8Hz); 6.78 (s, 1H), 6.62 (d, 1H, the research fund of Dankook University in 2015.
J=8.0Hz), 2.94 (t, 2H, J=6.8Hz), 1.95 (t, 2H, J=6.8Hz), 1.41
(s, 6H); 13C-NMR (100MHz, pyridine-d5) δ: 182.7, 165.9,
Conflict of Interest The authors declare no conflict of
161.8, 161.0, 147.1, 134.1, 134.1, 124.4, 123.0, 117.1, 117.1, interest.
114.3, 114.2, 112.3, 106.2, 76.6, 31.3, 26.6, 26.6, 16.0; LR-MS
(FAB) m/z 323 (M+H+); HR-MS (FAB) Calcd for C20H19O4
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Bromide (MTT) Assay Cell viability was determined by
MTT assay. Raw264.7 Macrophages were incubated with the
damaurone D (1) at different concentrations for 24h in 96-well
culture plates. After incubation, MTT (0.5mg/mL in phos-
phate buffered saline (PBS)) 20µL was added to each well and
the cells were further incubated for 4h at 37°C. The formation
of violet precipitate formazan was monitored at a wavelength
of 560nm and 670nm with spectrophotometer.
Treatment of Compounds Raw264.7 Macrophages were
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centrations for 1h and then treatment with LPS 10ng/mL for
24h in 12-well cell culture plate.
Western Blot Cells were harvest with lysis buffer
containing 10mM Tris–HCl (pH 7.1), 100mM NaCl, 1mM
ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 0.5%
Triton X-100, 0.5% Nonidet P-40, 1mM dithiothreitol and
0.5mM phenylmethylsulfonyl fluoride, supplemented with
inhibitors of proteinase and phosphatase. The protein con-
centrations in the cell lysates were determined by using a
Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA,

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