Biological evaluation of isoegomaketone isolated from perilla frutescens and its synthetic derivatives as anti-inflammatory agents

Article abstract of DOI:10.1007/s12272-011-0806-8

The anti-inflammatory activities of a prepared isoegomaketone 3a and its derivatives 3b-3f were evaluated in RAW 264.7 cells. Among these, the compound 3d was displayed the most potent inhibitory activities against production of nitric oxide, monocyte chemoattractant protein- 1 and interleukin-6. Based on these results, the abilities of compounds 3a-3f to modulate NF-κB and AP-1-mediated gene transcription using a luciferase reporter assay were investigated. The transcriptional activities of NF-κB and AP-1 decreased when pretreated with 3a- 3f. Interestingly, at 10 μM, compound 3d markedly suppressed the lipopolysaccharide-induced NF-κB and activator protein-1 DNA binding activities. Some preliminary structure-activity relationships were proposed that may provide a direction for further study.

Full text of DOI:10.1007/s12272-011-0806-8

Arch Pharm Res Vol 34, No 8, 1277-1282, 2011
DOI 10.1007/s12272-011-0806-8
Biological Evaluation of Isoegomaketone Isolated from Perilla frute-
scens and its Synthetic Derivatives as Anti-inflammatory Agents
Yong Dae Park1, Chang Hyun Jin1, Dae Seong Choi1, Myung-Woo Byun2, and Il Yun Jeong1
1Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongup 580-185, Korea and
2Department of Culinary Nutrition, Woosong University, Daejeon 300-718, Korea
(Received Januray 3, 2011/Revised March 22, 2011/Accepted April 4, 2011)
The anti-inflammatory activities of a prepared isoegomaketone 3a and its derivatives 3b-3f
were evaluated in RAW 264.7 cells. Among these, the compound 3d was displayed the most
potent inhibitory activities against production of nitric oxide, monocyte chemoattractant pro-
tein-1 and interleukin-6. Based on these results, the abilities of compounds 3a
NF- B and AP-1-mediated gene transcription using a luciferase reporter assay were investi-
gated. The transcriptional activities of NF- B and AP-1 decreased when pretreated with 3a
3f. Interestingly, at 10 M, compound 3d markedly suppressed the lipopolysaccharide-induced
NF-
-3f to modulate
κ
κ
-
µ
κ
B and activator protein-1 DNA binding activities. Some preliminary structure-activity
relationships were proposed that may provide a direction for further study.
Key words: Isoegomaketone, Anti-inflammatory activity, Synthesis, Perilla frutescens
tant protein-1 (MCP-1), inducible nitric oxide synthase,
cyclooxygenase-2, and intercellular adhesion mole-
Inflammation is a natural biological response to cule-1 (Aderem et al., 2000). NF- B is located in the
injury or infection in the human body. Unregulated cytoplasm of unstimulated cells in complex with I
inflammation results in the cause of a large number of which causes it to remain in its inactive form. Extra-
diseases including rheumatoid arthritis, obesity, car- cellular stimuli, such as lipopolysaccharide LPS),
diovascular diseases, neurodegenerative diseases, activate NF- B by inducing serine phosphorylation of
diabetes, and cancer. Much progress has been made in , which leads to its ubiquitination and degrada-
INTRODUCTION
κ
κBα,
(
κ
IκBα
the delineation of cell-signalling pathways that the tion. AP-1 is a transcription factor which is a homo- or
inflammation initiates a cascade of events that result heterodimeric protein composed of Fos family (c-Fos,
in the overproduction of certain inflammation-asso- Fos-B, Fra-1 and Fra-2) and Jun subfamily (c-Jun, Jun-
ciated genes and pro-inflammatory cytokines (O’Neill, B and Jun-D) proteins (Shaulian and Karin, 2002).
2006). Two inflammation-associated genes, nuclear
Inhibition of inflammatory cytokine and mediator
factor-
κ
-binding (NF- B) and activator protein-1 (AP- production serves as a key mechanism in the control
κ
1), control the production of the cytokines and inflam- of inflammation. A number of anti-inflammatory drugs
matory mediators elevated in inflammatory diseases that suppress single inflammatory mediator produc-
(Manning and Mercurio, 1997; Kristof et al., 2001). tion, such as IL-1, tumor necrosis factor-
α, or nitric
NF- B plays a pivotal role in the expression of various oxide (NO) have already entered clinical trials for the
κ
inducible target genes related to immune and inflam- treatment of inflammatory disorder (Reinhart and
matory responses. These genes include tumor necrosis Karzai, 2001). Drugs that suppress the expression of
factor-
α
, interleukin-6 (IL-6), monocyte chemoattrac- these inflammatory mediator productions have, there-
fore, attracted significant interest as potential thera-
Correspondence to: Il Yun Jeong, Advanced Radiation Technol-
ogy Institute, Korea Atomic Energy Research Institute, Jeongup
580-185, Korea
Tel: 82-63-570-3150, Fax: 82-63-570-3159
E-mail: iyjeong@kaeri.re.kr
peutics for the treatment of inflammatory diseases.
While searching for small molecule inhibitors of in-
flammatory mediator production in mouse macrophages
stimulated with LPS, we found that Perilla frutescens
1277

1278
Y. D. Park et al.
methanol extract inhibited NO production. The in-
vestigation of P. frutescens extract led to the isolation
of isoegomaketone (Park et al., 2010), which is an
General procedure for synthesis of isoegoma-
ketone (3a) and its derivatives 3b-f
To 10 mmol of the trans-enamino ketone 2a-2f in
essential oil component of P. frutescens. A previous 100 mL of dry tetrahydrofuran under nitrogen was
study from our laboratory demonstrated that the added 18 mL of 0.7 M isopropyllithium reagent in
inhibition mechanisms of LPS-induced NO production pentane. After stirring at
is distinct, in which isoegomaketone inhibited the solution was stirred to room temperature for 30 min
−
40oC for 30 min, the
activation of NF-
2010).
κ
B and AP-1 pathways (Jin et al., and 5 mL of water was added. The solvent was
evaporated in vacuo, and the residue was extracted
Chemical modification of isoegomaketone (3a), with dichloromethane. The organic layer was washed
focusing on the aromatic heterocyclic ring, was carried with saturated sodium chloride solution, dried over
out to improve the suppressive effects on the pro- MgSO4, and evaporated under reduced pressure. The
duction of NO, MCP-1, and IL-6. In this report, we obtained residue was purified by chromatography on
describe the synthesis, structure-activity relation- silica gel using n-hexane/ethyl acetate 9:1 to give the
ships, and biological activities of the novel series of compounds 3a-f
.
isoegomaketone derivatives.
(E)-3-(dimethylamino)-1-(furan-3-yl)prop-2-en-1-
one (2a)
Yield 57%; IR (
MATERIALS AND METHODS
General experimental procedures
ν
max, cm−1, KBr): 1640 (C=O); HRMS
1
calcd for C9H11NO2: 165.0789. Found: 165.0792; H-
1H-NMR and 13C-NMR were measured downfield NMR (500 MHz, CDCl3,
δ
, ppm): 2.89 (3H, s), 3.12
= 12.6 Hz), 6.76 (1H, d, = 1.7
= 1.1 Hz), 7.75 (1H, d, = 12.6 Hz),
= 1.7 Hz); 13C-NMR (125 MHz, CDCl3,
relative to tetramethylsilane in CDCl3 unless other- (3H, s), 5.40 (1H, d,
wise noted (value in ppm); coupling constants are Hz), 7.40 (1H, d,
reported in hertz, and conducted on a JNM-ECA 500 7.93 (1H, d,
J
J
J
J
J
J
δ,
spectrometer (Jeol). IR spectra (KBr) were recorded ppm): 36.9, 43.9, 93.1, 109.2, 129.2, 143.3, 144.7,
with a Bruker-Vector22 instrument (Bruker). High 153.2, 182.7.
resonance mass spectra (HRMS) were recorded on
JMS-700 (Jeol) and measured at Gyeongsang National
(E)-3-(dimethylamino)-1-(2,5-dimethylfuran-3-yl)
University (Chinju). The stationary phases used for prop-2-en-1-one (2b)
column chromatography (Silica gel 60, 70-230 mesh), Yield 68%; IR (
and TLC plates (Silical-gel 60 F254) were purchased calcd for C11H15NO2: 193.1128. Found: 193.1102; H-
ν
max, cm−1, KBr): 1641 (C=O); HRMS
1
form Merck KGaA. Spots were detected under UV NMR (500 MHz, CDCl3,
radiation. All the reagent-grade chemicals were pur- (3H, s), 2.90 (3H, s), 3.12 (3H, s), 5.35 (1H, d,
δ
, ppm): 2.24 (3H, s), 2.55
= 12.6
= 12.6 Hz); 13C-NMR
, ppm): 13.2, 14.1, 36.7, 43.8, 94.3,
105.8, 122.9, 149.0, 152.8, 154.6, 184.8.
J
chased from the Sigma-Aldrich.
Hz), 6.16 (1H, s), 7.67 (1H, d,
(125 MHz, CDCl3,
J
δ
General procedure for synthesis of 2-substi-
tuted-(
vatives 2a-f
E)-3-(dimethylamino)but-2-en-1-one deri-
(E)-3-(dimethylamino)-1-(furan-2-yl)prop-2-en-1-
A mixture of 20 mmol of acetylated 5-numbered one (2c)
aromatic heterocyclic compounds 1a-1f and 25 mL of Yield 61%; IR (
ν
max, cm−1, KBr): 1637 (C=O); HRMS
1
N
,N-dimethylformamide dimethyl acetal was heated calcd for C9H11NO2: 165.0789. Found: 165.0793; H-
under reflux for 12 h. Standard workup procedures NMR (500 MHz, CDCl3,
were follows. The mixture was evaporated in vacuo (3H, s), 5.67 (1H, d, = 12.6 Hz), 6.47 (1H, m), 7.06
and the residue was partitioned between the ethyl (1H, m), 7.48 (1H, m), 7.78 (1H, d,
= 12.6 Hz); 13C-
acetate and water. The organic extracts were combined NMR (125 MHz, CDCl3, , ppm): 37.2, 44.9, 91.4,
δ, ppm): 2.91 (3H, s), 3.13
J
J
δ
and washed in the indicated order using the following 111.7, 113.2, 144.1, 153.4, 154.7, 177.4.
aqueous solutions: water, 5% aqueous sodium carbonate
(NaHCO3) solution, and saturated sodium chloride
(NaCl) solution. Extracts were dried over anhydrous 2-en-1-one (2d)
magnesium sulfate (MgSO4), filtered, and evaporated Yield 67%; IR (
(E)-3-(dimethylamino)-1-(5-methylfuran-2-yl)prop-
ν
max, cm−1, KBr): 1644 (C=O); HRMS
1
in vacuo. Chromatographic separations were carried calcd for C10H13NO2: 179.0946. Found: 179.0943; H-
out on silica gel using chloroform/methanol 9:1 to give NMR (500 MHz, CDCl3,
δ
, ppm): 2.37 (3H, s), 2.92
compound 2a-2f (3H, s), 3.12 (3H, s), 5.62 (1H, d,
.
J
= 12.6 Hz), 6.09

Biological Evaluation of Isoegomaketone and its Synthetic Derivatives
1279
(1H, d,
J
= 3.4 Hz), 6.96 (1H, d,
J
= 3.4 Hz), 7.76 (1H, 7.62 (1H, m); 13C-NMR (125 MHz, CDCl3,
δ
, ppm):
d, δ, ppm): 21.1, 31.2, 112.1, 117.3, 121.5, 146.3, 153.2, 154.9,
J
= 12.6 Hz); 13C-NMR (125 MHz, CDCl3,
13.9, 36.8, 43.9, 91.3, 108.3, 114.9, 153.1, 153.3, 154.9, 178.3.
177.3.
(E)-4-methyl-1-(5-methylfuran-2-yl)pent-2-en-1-one
(
E
)-3-(dimethylamino)-1-(thiophen-3-yl)prop-2-en-
(3d)
Yield 73%; IR (
1-one (2e)
Yield 56%; IR (
ν
max, cm−1, KBr): 1659 (C=O); HRMS
1
ν
max, cm−1, KBr): 1641 (C=O); HRMS calcd for C11H14O2: 178.0993. Found: 179.0994; H-
1
calcd for C9H11NOS: 181.0561. Found: 181.0563; H- NMR (500 MHz, CDCl3,
δ, ppm): 0.96 (6H, d,
J
= 6.9
NMR (500 MHz, CDCl3,
(3H, s), 5.58 (1H, d, = 12.6 Hz), 7.27 (1H, dd,
5.1 Hz), 7.53 (1H, dd, = 1.1, 4.8 Hz), 7.77 (1H, d,
12.6 Hz), 7.90 (1H, dd,
= 1.1, 2.9 Hz); 13C-NMR (125 13.7, 21.1, 30.9, 108.8, 119.2, 121.8, 151.8, 153.9,
MHz, CDCl3, , ppm): 36.7, 43.8, 92.9, 125.4, 127.3, 157.6, 177.4.
128.4, 144.9, 153.9, 182.9.
δ
, ppm): 2.91 (3H, s), 3.13 Hz), 2.25 (3H, s), 2.38 (1H, m), 6.03 (1H, m), 6.54 (1H,
= 3.1, dd, = 1.4, 15.4 Hz), 6.92 (1H, dd, = 6.9, 15.4 Hz),
7.02 (1H, m); 13C-NMR (125 MHz, CDCl3,
, ppm):
J
J
J
J
J
J
=
δ
J
δ
(E)-4-methyl-1-(thiophen-3-yl)pent-2-en-1-one (3e)
(
E
)-3-(dimethylamino)-1-(1-methyl-1H-pyrrol-3-yl) Yield 28%; IR (
ν
max, cm−1, KBr): 1657 (C=O); HRMS
1
prop-2-en-1-one (2f)
calcd for C10H12OS: 180.0608. Found: 180.0605; H-
Yield 49%; IR (
ν
max, cm−1, KBr): 1644 (C=O); HRMS NMR (500 MHz, CDCl3,
δ
, ppm): 1.11 (6H, d,
= 1.4, 15.5 Hz), 7.03
= 6.8, 15.5 Hz), 7.32 (1H, m), 7.57 (1H, m),
= 12.6 Hz), 6.54 (2H, m), 7.21 8.05 (1H, m); 13C-NMR (125 MHz, CDCl3,
, ppm): 21.4,
= 12.6 Hz); 13C-NMR (125 MHz, 31.4, 123.6, 126.2, 127.4, 131.9, 142.8, 155.2, 184.6.
, ppm): 36.3, 36.9, 92.9, 108.6, 122.2, 124.7,
127.4, 152.1, 184.2.
J = 6.8
1
calcd for C10H14N2O: 178.1106. Found: 178.1104; H- Hz), 2.53 (1H, m), 6.69 (1H, dd,
J
NMR (500 MHz, CDCl3,
(3H, s), 5.48 (1H, d,
(1H, s), 7.69 (1H, d,
CDCl3,
δ, ppm): 2.96 (6H, s), 3.66 (1H, dd, J
J
δ
J
δ
(E)-4-methyl-1-(1-methyl-1H-pyrrol-3-yl)pent-2-en-
1-one (3f)
Yield 34%; IR (
(
E
)-1-(furan-3-yl)-4-methylpent-2-en-1-one (3a)
ν
max, cm−1, KBr): 1660 (C=O); HRMS
1
Yield 38%; IR (
ν
max, cm−1, KBr): 1668 (C=O); HRMS calcd for C11H15NO: 177.1153. Found: 177.1155; H-
1
calcd for C10H12O2: 164.0837. Found: 164.0841; H- NMR (500 MHz, CDCl3,
δ, ppm): 1.07 (6H, d,
J
J
= 6.8
= 1.4,
NMR (500 MHz, CDCl3,
Hz), 2.53 (1H, m), 6.49 (1H, dd,
(1H, m), 7.03 (1H, dd,
= 6.9, 15.4 Hz), 7.45 (1H, m), 6.8, 15.5 Hz), 7.26 (1H, m); 13C-NMR (125 MHz,
8.05 (1H, m); 13C-NMR (125 MHz, CDCl3,
, ppm): CDCl3, , ppm): 21.4, 31.1, 36.5, 109.6, 123.3, 124.1,
δ
, ppm): 1.12 (6H, d,
J
= 6.9 Hz), 2.49 (1H, m), 3.67 (3H, s), 6.54 (1H, dd,
J
= 1.4, 15.4 Hz), 6.83 15.5 Hz), 6.56 (1H, m), 6.62 (1H, m), 6.94 (1H, dd, J =
J
δ
δ
21.3, 31.2, 109.1, 123.9, 128.1, 144.1, 147.1, 154.5, 126.2, 126.9, 152.2, 185.2.
184.8.
Cytotoxicity assay
(
E
)-1-(2,5-dimethylfuran-3-yl)-4-methylpent-2-en-
To measure the cell viability, an EZ-Cytox cell
viability assay kit (DAEIL lab) was used. The cells
1-one (3b)
Yield 62%; IR (
ν
max, cm−1, KBr): 1668 (C=O); HRMS were cultured in a 96-well plate (5.0 × 104 cell/well) at
calcd for C12H16O2: 192.1150. Found: 192.1149; H- 37oC, 5% CO2 for 24 h and subsequently treated with
1
NMR (500 MHz, CDCl3,
Hz), 2.26 (3H, s), 2.50 (1H, m), 2.55 (3H, s), 6.23 (1H, h. After the incubation period, 10
s), 6.47 (1H, dd, = 1.4, 15.4 Hz), 6.94 (1H, dd, solution was added into each well and incubated for 4
6.6, 15.4 Hz); 13C-NMR (125 MHz, CDCl3,
, ppm): h. The index of cell viability was determined by mea-
13.1, 14.3, 21.4, 31.1, 105.7, 122.1, 124.9, 149.8, 153.6, suring the OD of formazan production at 480 nm using
δ, ppm): 1.10 (6H, d,
J
= 6.6 various concentrations of the derivatives 3a-3f for 24
µL of the kit
J
J =
δ
157.5, 186.7.
an enzyme-linked immunosorbent assay (ELISA) plate
reader at an absorbance of 480 nm. The reference
wavelength was 650 nm.
(E)-1-(furan-2-yl)-4-methylpent-2-en-1-one (3c)
Yield 31%; IR (
calcd for C10H12O2: 164.0837. Found: 164.0836; H-
ν
max, cm−1, KBr): 1664 (C=O); HRMS
1
NO measurement
NMR (500 MHz, CDCl3,
Hz), 2.56 (1H, m), 6.55 (1H, m), 6.75 (1H, dd,
15.4 Hz), 7.13 (1H, dd, = 6.9, 15.4 Hz), 7.25 (1H, m), fetal bovine serum (FBS), penicillin (100 U/mL), and
δ, ppm): 1.12 (6H, d,
J
J
= 6.9
RAW 264.7 cells were cultured in Dulbecco’s modifi-
= 1.4, ed Eagle’s medium (DMEM) supplemented with 10%
J

1280
Y. D. Park et al.
streptomycin (100
µ
g/mL), and held at 37oC with 5% LPS at a concentration of 1
µg/mL for 24 h. Luciferase
CO2. The cells were cultured in a 96-well plate at a activity was measured with the Dual-luciferase Reporter
concentration of 2.0 × 104 cell/well for 24 h. Cells were Assay System (Promega).
then treated with various concentrations of the
derivatives 3a-3f for 2 h. Then, cells were treated with
LPS (1 g/mL) for 18 h. The cell supernatants were
collected at the end of the culture and tested for
nitrite, as a measurement of NO production. Equal synthesized according to the method shown in Scheme
volumes of the Griess reagent (100 L; Sigma-Aldrich) 1. The conversion of ketones to -unsaturated
were added to each cell supernatant (100 L), and the ketones was performed as previously reported method
absorbance was measured at 570 nm. The concentration (Abdulla and Fuhr, 1978). The appropriate commercially
of nitrite ( M) was calculated from a standard curve available acetylated aromatic heterocyclic compounds
drawn with a known concentration of sodium nitrite 1a and N,N-dimethylformamide dimethyl acetal
dissolved in DMEM. The results are presented as the were heated under reflux for 12 h to produce com-
mean ± S.D. from 3 separate experiments, with 4 re- pounds 2a . The isoegomaketone derivatives 3a
were obtained by isopropylation using an isopropylli-
RESULTS AND DISCUSSION
µ
Isoegomaketone 3a and its derivatives 3b-f were
µ
α,β
µ
µ
-f
-f
-f
plicates each.
thium reagent. The chemical structures of these com-
pounds were determined using several spectroscopic
Measurement of MCP-1 and IL-6 by ELISA
1
Cells cultured at a concentration of 2.0 × 105 cell/mL analyses including IR, H-NMR, 13C-NMR and mass
were incubated for 24 h and then the derivatives 3a- spectrometry.
3f were added for 2 h. Then, LPS (1
µg/mL) was added
The pro-inflammatory cytokine, IL-6, as well as pro-
for 4 h. To determine the resultant cytokines, the inflammatory mediators, NO and MCP-1, play impor-
supernatants were harvested and the levels of IL-6 tant roles in the inflammatory process (Heinrich et al.,
and MCP-1 were measured by using an ELISA kit 2003; Melgarejo et al., 2009). Therefore, the compounds
(R&D systems). The results are presented as the mean were evaluated for their suppressive activity against
± S.D. from 3 separate experiments, with 4 replicates NO, MCP-1, and IL-6 production using a murine
each.
macrophage cell line, RAW 264.7, induced by LPS. We
initially examined the cytotoxicity. After treatment
with compounds 3a-3f, RAW 264.7 cell viability was
Luciferase assay
The cells were cultured in a 6-well dish for 24 h at a determined using an MTT assay. Compounds 3a
-
M
3f
concentration of 4 × 105 cell/mL. Vectors were trans- did not significantly affect cell viability in the 0-10
µ
fected into the cell using Lipofectamine 2000 according range (Fig. 1). As shown in Table I, isoegomaketone
to the manufacturer's instructions. The pNF- B-Luc 3a showed moderate potency with an IC50 value of 6.9
(Stratagene) and pAP-1-Luc vectors (Panomics) were and 6.6 M against NO and MCP-1, respectively.
transfected into cells at concentration of 5 g per well. Introduction of a methyl group at the 2,5-positions of
The pRL-TK vector (Promega) was transfected into the furan ring 3b resulted in a two-fold increase in
cells at a concentration of 1 g per well. After the potency in suppression of NO production, but only
transfection, compounds 3a-3f were added at a 10 slightly improved inhibitory activity toward MCP-1
concentration for 2 h and the cells were treated with production (IC50 5.1 M). Compound 3c, which con-
κ
µ
µ
µ
µM
µ
Scheme 1. Synthesis of derivatives 3a-3f. Reagents and conditions: (a) (CH3)2NCH(OCH3)2, reflux; (b) i-PrLi, THF, -40.

Biological Evaluation of Isoegomaketone and its Synthetic Derivatives
1281
Fig. 2. Effect of derivatives 3a-3f on the NF-
pathway. After transfection of pNF-
vectors, the pRL-TK vector was transfected into cells. After
transfection, derivatives 3a-3f were added at a 10 µM for 2
h and the cells were treated with LPS for 24 h.
κ
B and AP-1
B-Luc or pAP-1-Luc
κ
Fig. 1. Cytotoxicity of derivatives 3a
RAW 264.7 cells were incubated with the derivatives 3a
for 24 h, and cell viability was determined by an MTT assay.
The absorbance of control cells was taken as 100%. The
results are expressed as the mean ± S.D. of values obtained
from triplicate cultures. Data shown are representative of
three independent experiments with similar results.
-
3f in RAW 264.7 cells.
-3f
study, we hypothesized that isoegomaketone deriva-
tives 3a-3f would inhibit the expression of these genes
through the suppression of NF-
κB or AP-1 activation.
Table I. Inhibitory activities (IC50) of derivatives 3a-3f
NF- B plays an important role in many aspects of
κ
against LPS-induced NO, MCP-1 and IL-6 production
human inflammatory diseases (Diaz-Guerra et al.,
1996). As indicated in Fig. 2, LPS treatment caused a
IC50 (µ
M) a
Compound
significant increase in NF-κB and AP-1 DNA binding
activities. These binding activities were decreased by
pretreatment with 3a-3f. In particular, compound 3d
markedly suppressed the LPS-induced NF-
NO
MCP-1
IL-6
3a
3b
3c
3d
3e
3f
6.9 ± 0.21
3.8 ± 0.31
5.5 ± 0.47
1.9 ± 0.18
8.2 ± 1.21
8.4 ± 0.84
6.6 ± 0.45
5.1 ± 0.18
4.1 ± 0.12
2.0 ± 0.34
14.8 ± 1.14
12.6 ± 0.78
13.3 ± 1.14
14.2 ± 1.21
> 15
19.4 ± 0.84
> 20
κB and
AP-1 DNA binding activities at 10 M. These results
µ
are strongly suggest that the inhibitory effect of iso-
egomaketone derivatives on LPS-stimulated pro-in-
flammatory cytokines and mediator production might
16.2 ± 0.89
aIC50 values are calculated from means ± S.D. of three ex-
periments.
be regulated by both NF-
activation.
κB and AP-1 transcriptional
In this study we have described the anti-inflam-
tains an α,β-unsaturated carbonyl side chain substitu- matory activities of isoegomaketone derivatives. To
tion at the 2-position of furan ring, was equipotent to investigate the mode of action of derivatives 3a-3f as
compound 3a. Introduction of a methyl group at the 5- anti-inflammatory agents, the effect on LPS-induced
position of compound 3d, however, resulted in a three- inflammatory responses was investigated in a RAW
fold improvement in inhibitory activities toward NO 264.7 mouse macrophage cell line. Among all the
and MCP-1 production, with IC50 values of 1.9 and 2.0 compounds prepared, 3d exhibited the most potent
µ
M respectively. These data suggest that hydrophobic inhibitory effect on the production of NO, MCP-1, and
substituents at the 2- and 5-position of furan ring are IL-6. These IC50 values were approximately 3-fold
favorable for its inhibitory effects. The thiophene-, and stronger than isoegomaketone. Furthermore, 3d showed
pyrrole-substituted analogues 3e-f at the 3-position of significant suppression of NF-
κB and AP-1 DNA bind-
the compounds showed slightly lower activities. In the ing activities. The -unsaturated ketone position of
α,β
case of cytokine IL-6, inhibitory activity was not signi- the isoegomaketone is most likely the pharmacophore
ficantly improved in these derivatives, but compound of this class of compounds since naturally occurring
3d showed slightly higher activity compared to com- perillaketone which is C-C double bond reduced com-
pound 3a, with an IC50 value of 9.4
Next, attention was focused on the ability of the effect on NO production (Jin et al., 2010).
compounds 3a 3f to modulate NF- B and AP-1-medi- In conclusion, we have discovered a novel series of
µM.
pound of isoegomaketone completely lost inhibitory
-
κ
ated gene transcription using a luciferase reporter isoegomaketone derivatives. A representative com-
assay in RAW 264.7 cells. For the purpose of this pound 3d of the series showed significant anti-in-

1282
Y. D. Park et al.
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ACKNOWLEDGEMENTS
This research was supported by Agricultural R&D
Promotion Center of the Ministry for Food, Agricul-
ture, Forestry and Fisheries, Korea.
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