Inhibitory effects of 6-alkoxycoumarin and 7-alkoxycoumarin derivatives on lipopolysaccharide/interferon γ-stimulated nitric oxide production in RAW264 cells

Article abstract of DOI:10.1248/bpb.35.963

Coumarin and its derivatives are well known for their anti-inflammatory and anti-oxidative effects. In this study, we synthesized 32 coumarin derivatives from commercially available 6-hydroxycoumarin (6HC) and 7-hydroxycoumarin (7HC) and examined their ef

Full text of DOI:10.1248/bpb.35.963

June 2012
Regular Article
Biol. Pharm. Bull. 35(6) 963–966 (2012)
963
Inhibitory Effects of 6-Alkoxycoumarin and 7-Alkoxycoumarin
Derivatives on Lipopolysaccharide/Interferon γ-Stimulated Nitric Oxide
Production in RAW264 Cells
,a
Morina Adfa,a,b Tomohiro Itoh,c Yosuke Hattori,a and Mamoru Koketsu*
a Department of Materials Science and Technology, Faculty of Engineering, Gifu University; 1–1 Yanagido, Gifu
b
501–1193, Japan: Department of Chemistry, Bengkulu University; Jl. Raya Kandang Limun Bengkulu 38371,
c
Indonesia: and Faculty of Agriculture, Kinki University; 3327–204 Nakamachi, Nara 631–8505, Japan.
Received February 24, 2012; accepted March 26, 2012
Coumarin and its derivatives are well known for their anti-inflammatory and anti-oxidative effects. In
this study, we synthesized 32 coumarin derivatives from commercially available 6-hydroxycoumarin (6HC)
and 7-hydroxycoumarin (7HC) and examined their effects on lipopolysaccharide/interferon γ (LPS/IFNγ)-
stimulated nitric oxide (NO) production in murine macrophage RAW264 cells. Among these derivatives,
6HC-8 (6-(3-phenylpropoxy)coumarin), 6HC-14 (6-(2-octynyloxy)coumarin), 7HC-14 (7-(2-octynyloxy)cou-
marin), and 7HC-16 (7-(3,5-dimethoxybenzyloxy)coumarin) markedly suppressed NO production at low con-
centration (25µM). These synthesized coumarin derivatives also markedly inhibited inducible NO synthase
(iNOS) protein and mRNA expression, as assessed by western blotting and quantitative real time-polymerase
chain reaction (RT-PCR).
Key words coumarin derivative; lipopolysaccharide; interferon-γ; nitric oxide; inducible nitric oxide syn-
thase; RAW264 cell
Nitric oxide (NO) is a free radical species that contributes macrophage RAW264 cells. To identify the mechanisms un-
to several important biological functions, such as host defense derlying the suppression of LPS/IFNγ-stimulated NO produc-
against pathogens, inhibition of tumor cells, and neurotrans- tion by these coumarin derivatives, we examined the protein
mission.1–4) However, excessive NO production can be harmful and mRNA expression levels of inducible NO synthase (iNOS)
and trigger rheumatoid arthritis, gastritis, bowel inflammation, using western blotting and quantitative real time-polymerase
neuronal cell death, and bronchitis.5,6) NO is synthesized by chain reaction (RT-PCR). Based on the results obtained, we
an inducible isoform of NO synthase (iNOS), which cata- propose that these synthesized coumarin derivatives suppress
lyzes the oxidation of the equivalent guanidine nitrogen of L- inflammatory responses in macrophages, at least partly by the
arginine.7–10) Macrophages produce large amount of NO after inhibition of iNOS expression.
stimulation with lipopolysaccharide (LPS), interferons (IFNs),
tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β).
Macrophages express CD14, CD11b/18, and toll-like recep-
tors (TLRs) on their cell surfaces.11–13) TLR4 is a well-known
MATERIALS AND METHODS
Synthesis of Coumarin Derivatives from 6HC and 7HC
signaling partner for LPS/CD14 interactions. LPS binds to 6-Hydroxycoumarin (6HC) was purchased from Sigma-
CD14/TLR4 that activates mitogen-activated protein kinases Aldrich and 7-hydroxycoumarin (7HC) was purchased from
(MAPKs) including extracellular signal-regulated kinase Tokyo Chemical Industry Co., Ltd. From these, we prepared
(ERK), c-Jun NH2-terminal protein kinase, and p38 MAP 32 derivatives using previously reported procedures.19) Their
kinase (p38). This leads to the expression of iNOS, cyclooxy- chemical structures were confirmed by nuclear magnetic
genase-2 (COX-2), TNF-α, and interferon β (IFNβ).14,15) On the resonance (NMR) and mass spectrometric analyses. In brief,
contrary, IFNβ and IFNγ bind to type I and type II IFN recep- a mixture of 6HC or 7HC (30mg; 0.19mmol), an alkyl or
tors, respectively, expressed on the surfaces of macrophages, acyl halide (0.38mmol), and potassium carbonate (79mg;
and activate Janus kinase (JAK)-signal transducers and activa- 0.57mmol) in N,N-dimethyl formamide (DMF) (5mL) was
°
tors of transcription (STAT) signaling. This results in the up- stirred at 55–85 C for times ranging from 0.5h until over-
regulation of IFN regulatory factor 1 (IRF1).16) Both IRF1 and night. The reaction mixture was extracted with ethyl acetate
STAT1 bind to the 5′-flanking region of the iNOS promoter and the combined extracts were washed with water. The
and enhance NO production. LPS also activates other signal- organic layer was dried over sodium sulfate and evaporated
ing molecules, including phosphoinositide-3 kinase, protein to dryness. The residue was then purified by column chro-
kinase C, and small-molecular weight GTPases such as Rac matography on silica gel with n-hexane/ethyl acetate (7:3) to
and CDC42.17,18) Thus, modulation of LPS/IFNγ signaling can yield the products 6HC-1–6HC-8, 6HC-10–6HC-16, 7HC-
alter iNOS expression and NO production.
1–7HC-8, and 7HC-10–7HC-16. Instead of K2CO3, Et3N
In this study, 32 coumarin derivatives were synthesized (79µL; 0.57mmol) and DMAP (23.2mg; 0.19mmol) were used
°
from commercially available 6-hydroxycoumarin (6HC) to prepare 6HC-9 and 7HC-9 at 80 C for 1h (Chart 1). There-
and 7-hydroxycoumarin (7HC). Some of these derivatives fore, we prepared 32 coumarin derivatives and examined their
suppressed LPS/IFNγ-stimulated NO production in murine inhibitory effects on LPS/IFNγ-stimulated NO production in
RAW264 cells. The physical and spectroscopic characteristics
the most active compounds were as follows.
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: koketsu@gifu-u.ac.jp
© 2012 The Pharmaceutical Society of Japan

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Vol. 35, No. 6
(C-4), 155.8 (C-9), 161.1 (C-2), 161.3 (C-7); FAB-MS m/z: 271
[M+H]+.
7-(3,5-Dimethoxybenzyloxy)coumarin (7HC-16): Colorless
1
°
crystals, mp 115 C (83% yield): H-NMR (400MHz, CDCl3)
δ: 3.80 (6H, s, 2CH3), 5.07 (2H, s, H-1′), 6.25 (1H, d, J=9.6Hz,
H-3), 6.43 (1H, s, H-Ar), 6.57 (2H, s, H-Ar), 6.86 (1H, s, H-8),
6.91 (1H, d, J=8.7Hz, H-6), 7.37 (1H, d, J=8.7Hz, H-5), 7.63
(1H, d, J=9.6Hz, H-4); 13C-NMR (100MHz, CDCl3) δ: 55.5
(2CH3), 70.5 (C-1′), 100.2 (C-4 Ar), 102.1 (C-8), 105.3 (C-2 Ar,
C-6 Ar), 112.9 (C-10), 113.3 (C-6, C-3), 128.9 (C-5), 138.2 (C-1
Ar), 143.5 (C-4), 155.9 (C-9), 161.2 (C-3 Ar, C-5 Ar), 161.9 (C-
2), 161.9 (C-7); FAB-MS m/z: 313 [M+H]+.
Cell Culture and Treatment Murine macrophage
RAW264 cells were purchased from RIKEN Bio Resource
’
Center (Tsukuba, Japan). Cells were cultured in Dulbecco s
’
modified Eagle s medium (DMEM) containing 10% heat-in-
activated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA,
U.S.A.), 100U/mL of penicillin and 100µg/mL streptomycin
°
in a humidified atmosphere of 5% CO2 at 37 C. RAW264 cells
(1×105 cells/well) were seeded into 24-well micro plates and
cultured for 12h. After incubation, cells were pre-treated with
each synthesized coumarin derivative for 2h. The treated cells
were then co-stimulated with or without LPS (final concentra-
tion: 200ng/mL) (Sigma-Aldrich) and IFNγ (final concentra-
tion: 25ng/mL) (Millipore, Bedford, MA, U.S.A.).
Chart 1. Synthesis of 6-Alkoxycoumarin (6HC-1–6HC-16) and 7-Al-
koxycoumarin Derivatives (7HC-1–7HC-16).
Measurement of NO Production Cell culture media
°
6-(3-Phenylpropoxy)coumarin (6HC-8): Colorless crystals, were centrifuged at 4 C for 5min. A Griess reagent kit (Pro-
1
°
mp 84–85 C (98% yield): H-NMR (600MHz, CDCl3) δ: 2.13 mega, Madison, WI, U.S.A.) was used to measure the amount
(2H, quint., J=6.2Hz, H-2′), 2.83 (2H, t, J=7.6Hz, H-3′), 3.98 of nitrite, a stable metabolite of NO, in the supernatants.
(2H, t, J=6.2Hz, H-1′), 6.42 (1H, d, J=9.6Hz, H-3), 6.88 (1H,
Western Blotting Whole cell extracts were prepared by
d, J=2.8Hz, H-5), 7.10 (1H, dd, J=9.6, 2.8Hz, H-7), 7.20–7.31 lysing cells in RIPA buffer containing a complete protease
(6H, m, Ar-H, H-8), 7.63 (1H, d, J=9.6Hz, H-4); 13C-NMR inhibitor cocktail and a phosphatase inhibitor cocktail (Roche,
(150MHz, CDCl3) δ: 30.8 (C-2′), 32.2 (C-3′), 67.7 (C-1′), 110.9 Penzberg, Germany). Samples were subjected to sodium do-
(C-5), 117.2 (C-8), 118.0 (C-3), 119.3 (C-10), 120.1 (C-7), 126.2 decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
(C-3 Ar, C-5 Ar), 128.61 (C-4 Ar), 128.64 (C-2 Ar, C-6 Ar), and electroblotted onto PVDF membranes. Membranes were
141.3 (C-1 Ar), 143.4 (C-4), 148.5 (C-9), 155.6 (C-6), 161.1 (C- incubated with a primary antibody, followed by incubation
2); FAB-MS m/z: 281 [M+H]+; Anal. Calcd for C18H16O3: C, with a horseradish peroxidase-conjugated secondary antibody.
77.12; H, 5.75. Found: C, 77.24; H, 6.09.
Immunolabeled proteins were detected using an ECL chemilu-
6-(2-Octynyloxy)coumarin (6HC-14): Pale yellow crys- minescence kit (GE Healthcare, Piscataway, NJ, U.S.A.) and a
tals, mp 47 C (89% yield): 1H-NMR (400MHz, CDCl3) δ: LAS-4000 lumino-image analyzer (FUJIFILM, Tokyo, Japan).
°
0.86 (3H, t, J=7.2Hz, H-8′), 1.21–1.37 (4H, m, H-7′, H-6′),
Quantitative RT-PCR Total RNA was extracted from
1.43–1.54 (2H, m, H-5′), 2.16–2.34 (2H, m, H-4′), 4.72 (2H, cells using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.)
t, J=1.8Hz H-1′), 6.43 (1H, d, J=9.4Hz, H-3), 7.03 (1H, d, followed by DNase I treatment. cDNA was synthesized from
J=2.7Hz, H-5), 7.18 (1H, dd, J=9.0, 2.7Hz, H-7), 7.27 (1H, 0.25mg of total RNA using a PrimeScript reagent kit (Ta-
d, J=9.0Hz, H-8), 7.67 (1H, d, J=9.4Hz, H-4); 13C-NMR KaRa Bio, Shiga, Japan). cDNA was subjected to quantitative
(100MHz, CDCl3) δ: 14.1 (C-8′), 18.8 (C-4′), 22.2 (C-7′), 28.2 RT-PCR using a Thermal Cycler Dice real-time PCR system
‘
(C-5′), 31.1 (C-6′), 57.2 (C-1′), 74.4 (C-2′), 89.3 (C-3′), 111.9 (TP800, TaKaRa Bio). Primers for iNOS and glyceraldehyde-
’
(C-5), 117.2 (C-3), 117.9 (C-8), 119.2 (C-10), 120.4 (C-7), 143.3 3-phosphate dehydrogenase (GAPDH) were purchased from
(C-4), 148.9 (C-9), 154.3 (C-6), 161.1 (C-2); FAB-MS m/z: 271 TaKaRa Bio. The expression level of each gene was deter-
[M+H]+; Anal. Calcd for C17H18O3: C, 75.53; H, 6.71. Found: mined using the comparative Ct method and normalized to
‘
’
C, 75.29; H, 7.11.
that of GAPDH, an internal control. The PCR reaction con-
°
°
°
7-(2-Octynyloxy)coumarin (7HC-14): Pale yellow pow- sisted of 45 cycles (95 C for 10s, 60 C for 40s, and 72 C for
der, mp 59 C (95% yield): 1H-NMR (400MHz, CDCl3) δ: 1s) after an initial denaturation step (95 C for 10min).
0.86 (3H, t, J=7.2Hz, H-8′), 1.23–1.40 (4H, m, H-6′, H-7′), Statistical Analysis All data were analyzed using Stu-
°
°
’
1.44–1.55 (2H, m, H-5′), 2.17–2.25 (2H, m, H-4′), 4.74 (2H, dent s t-test. The differences were considered significant at
t, J=1.8Hz, H-1′), 6.26 (1H, d, J=9.4Hz, H-3), 6.90 (1H, dd, p<0.01.
J=8.5, 2.3Hz, H-6), 6.94 (1H, d, J=2.3Hz, H-8), 7.38 (1H,
d, J=8.5Hz, H-5), 7.64 (1H, d, J=9.4Hz, H-4); 13C-NMR
RESULTS AND DISCUSSION
(100MHz, CDCl3) δ: 14.0 (C-8′), 18.8 (C-4′), 22.3 (C-7′), 28.1
(C-5′), 31.1 (C-6′), 57.1 (C-1′), 73.8 (C-2′), 89.7 (C-3′), 102.2
As shown in Fig. 1, synthesized coumarin de-
(6HC-5–6HC-10, 6HC-14, 7HC-5–7HC-7,
(C-8), 113.0 (C-10), 113.4 (C-6), 113.5 (C-3), 128.8 (C-5), 143.5 rivatives

June 2012
965
Fig. 1. Effects of Synthesized Coumarin Derivatives (6HC-1–6HC-16, 7HC-1–7HC-16) on LPS/IFNγ-Stimulated NO Release in RAW264 Cells
RAW264 cells were pretreated with or without the each synthesized coumarin derivative (25 or 50µM) for 2h. The cells were then stimulated with or without LPS
(200ng/mL)/IFNγ (25ng/mL) for 24h. After 24h later, cell culture media were harvested for measurement of nitrite, a stable metabolite of NO (mean S.E., n=9). Aster-
’
isks indicate statistical significance as determined by Student s t-test (*p<0.01).
Fig. 2. Effects of Synthesized Coumarin Derivatives (6HC-6, 6HC-7, 6HC-8, 6HC-10, 6HC-14, 6HC-16, 7HC-6, 7HC-7, 7HC-10, 7HC-14, and
7HC-16) on Expression of iNOS in LPS/IFNγ-Stimulated RAW264 Cells
RAW264 cells were pretreated with or without the each synthesized coumarin derivative (25µM) for 2h. The cells were then stimulated with or without LPS (200ng/
mL)/IFNγ (25ng/mL) for 24h. (A) After 24h later, the cell lysates were harvested and subjected to Western blot analysis for iNOS, and β-actin. A representative blot from
three independent experiments is shown. (B) After 12h later, total RNA was harvested from the each cell and subjected to quantitative RT-PCR for iNOS (mean S.E.,
’
n=3). Asterisks indicate statistical significance as determined by Student s t-test (*p<0.05, **p<0.01).
7HC-14, and 7HC-16) could suppress LPS/IFNγ-stimulated position of the coumarin skeleton exhibited good inhibitory
NO production in RAW264 cells. Among these, 6HC–8 effects on LPS/IFNγ-stimulated NO production in RAW264
(6-(3-phenylpropoxy)coumarin), 6HC–14 (6-(2-octynyloxy)- cells. On comparison, 3-phenylpropoxyl (7HC-8) and 3,5-di-
coumarin), 7HC–14 (7-(2-octynyloxy)coumarin), and 7HC–16 methoxybenzyloxyl (6HC-16) groups at the C-7 and C-6 posi-
(7-(3,5-dimethoxybenzyloxy)coumarin) markedly suppressed tions of the coumarin skeleton exhibited no inhibitory effects.
NO production at low concentration (25µM). In contrast, other
To clarify the mechanisms underlying the inhibitory ef-
synthesized coumarin derivatives exhibited lower inhibitory fects of these synthesized coumarin derivatives on LPS/IFNγ-
effects or were inactive. Interestingly, 6HC-8 with a 3-phen- stimulated NO production, we first examined the iNOS pro-
ylpropoxyl group at the C-6 position of the coumarin skeleton tein expression levels by western blotting. The results in Fig.
and 7HC-16 with a 3,5-dimethoxybenzyloxyl group at the C-7 2A show that compared to RAW264 cells treated only with

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inflammatory bowel disease. Aliment. Pharmacol. Ther., 20 (Suppl.
4), 79–83 (2004).
LPS/IFNγ, iNOS protein expression levels were significantly
suppressed after treatment with the synthesized coumarin de-
rivatives (6HC-6, 6HC-7, 6HC-8, 6HC-10, 6HC-14, 7HC-6,
7HC-7, 7HC-10, 7HC-14, and 7HC-16). We further examined
iNOS mRNA expression levels using quantitative real time-
PCR analysis. The iNOS mRNA expression levels were also
significantly suppressed by these synthesized derivatives (Fig.
2B). These results suggested that the synthesized coumarin
derivatives down regulated iNOS expression at the transcrip-
tional level.
Kim et al. reported that decursin, derived from the roots of
Angelica gigas with a coumarin skeleton, suppressed inflam-
matory responses in RAW264 cells through the inactivation
of nuclear factor-kappaB (NF-κB).20) Upon LPS stimulation,
TLR4 signaling activates transcription factors, such as NF-
κB, activator protein 1 (AP1), and ELK1, culminating in the
expression of pro-inflammatory genes, including those for
iNOS, cyclooxygenase 2 (COX2), TNFα, and IFNβ. Thus,
inactivation of NF-κB by phytochemicals is an effective anti-
inflammatory mechanism. Further studies will be required to
clarify the inhibitory mechanisms of iNOS mRNA expression
by these synthesized coumarin derivatives.
In conclusion, the present study indicates that synthesized
coumarin derivatives from commercially available 6HC and
7HC, including 6HC–8 (6-(3-phenylpropoxy)coumarin), 6HC–
14 (6-(2-octynyloxy)coumarin), 7HC–14 (7-(2-octynyloxy)-
coumarin), and 7HC–16 (7-(3,5-dimethoxybenzyloxy)-
coumarin), could suppress LPS/IFNγ-stimulated NO produc-
tion in murine macrophage RAW 264 cells. This inhibitory
effect was mediated by modulating the transcriptional ma-
chinery of iNOS mRNA. Our findings suggest that these
synthesized coumarin derivatives could be effective agents for
alleviating symptoms of inflammation.
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