Synthesis and anti-inflammatory effects of a series of novel 7-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.052

A number of 7-hydroxycoumarins have been synthesised by Pechmann cyclisation using differently substituted resorcinols employing perchloric acid as the condensing agent. All the compounds have been characterised by analytical and spectroscopic methods. The anti-inflammatory properties were tested with LPS-induced inflammation in J774 macrophages. Expression of iNOS and COX-2 was determined by Western blot, NO by nitrite assay and IL-6 by ELISA analyses. Fifteen of the tested 7-hydroxycoumarins also inhibited IL-6 production but none of them had any major inhibitory effect on COX-2 expression.

Full text of DOI:10.1016/j.ejmech.2011.05.052

European Journal of Medicinal Chemistry 46 (2011) 3845e3850
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anti-inflammatory effects of a series of novel 7-hydroxycoumarin
derivatives
,
Juri M. Timonen a, Riina M. Nieminen b, Outi Sareila b, Antonis Goulas b c, Lauri J. Moilanen b,
,
Matti Haukka a, Pirjo Vainiotalo a, Eeva Moilanen b, Paula H. Aulaskari a
*
a Department of Chemistry, University of Eastern Finland, Joensuu Campus, P.O. Box 111, FIN-80101 Joensuu, Finland
b The Immunopharmacology Research Group, Medical School, University of Tampere and Tampere University Hospital, Tampere, Finland
c Department of Pharmacology, School of Medicine, Aristotle University of Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 7-hydroxycoumarins have been synthesised by Pechmann cyclisation using differently
substituted resorcinols employing perchloric acid as the condensing agent. All the compounds have been
characterised by analytical and spectroscopic methods. The anti-inflammatory properties were tested
with LPS-induced inflammation in J774 macrophages. Expression of iNOS and COX-2 was determined by
Western blot, NO by nitrite assay and IL-6 by ELISA analyses. Fifteen of the tested 7-hydroxycoumarins
also inhibited IL-6 production but none of them had any major inhibitory effect on COX-2 expression.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 24 January 2011
Received in revised form
19 May 2011
Accepted 22 May 2011
Available online 30 May 2011
Keywords:
Coumarin
von Pechmann synthesis
Nitric oxide
Inducible nitric oxide synthase
Interleukin-6
Anti‐inflammatory drugs
1. Introduction
synthesised twenty-two 7-hydroxycoumarin derivatives and
investigated systematically their anti-inflammatory effects on the
Coumarins, also known as 2H-benzo-1-pyran-2-ones, are
naturally occurring heterocyclic compounds that contain oxygen
[1]. In the literature, some coumarin derivatives have been reported
to inhibit NO production [2,3]. Further studies have shown some
expression of inflammatory enzymes iNOS and COX-2, and on the
production of inflammatory factors NO and IL-6 in activated
macrophages.
plant derived coumarins to inhibit the activation of NF-kB [4]. This
2. Chemistry
transcriptional factor is known to bind to the promoter region of
iNOS gene and to play a pivotal role in LPS mediated iNOS
expression in macrophages and other cells.
A few studies on the synthesis of 7-hydroxycoumarins have
been reported [5,6], and these served as a starting point for our
more extensive approach. Some 7-methoxycoumarins have been
reported to inhibit the iNOS e NO pathway [7]. Coumarins are
metabolised to 7-hydroxycoumarins in man [8] and reported
activities of different coumarins may be partially related to their
metabolites.
A sulphuric acid or perchloric acid catalysed von Pechmann
reaction is a common method to prepare 4-substituted coumarins
in a laboratory scale [5]. Knoevenagel, Wittig and Perkin reactions
have also been used [6,9]. The differences in reactions are obvious if
we examine the starting materials for them. In Knoevenagel, Wittig
and Perkin reactions, salicylaldehyde with b-ketoester, triphenyl-
phosphine derivative or anhydride, respectively, have been used as
starting materials, while in the von Pechmann reaction, substituted
phenols and b-ketoesters serve as starting materials. In commercial
In this study, we focused on 7-hydroxycoumarins because it can
help to design new pro-drugs based on coumarin skeleton. We
use, the Knoevenagel and Perkin reactions are used more often
than the von Pechmann reaction.
In the literature, reactions in perchloric acid and at a tempera-
ture above 80 ꢀC have been left to proceed for 3e5 h [5]. Experi-
ments have also been carried out in sulphuric acid, in which the
* Corresponding author. Tel.: þ358 13 2513365; fax: þ358 13 2513390.
E-mail address: paula.aulaskari@uef.fi (P.H. Aulaskari).
addition of b
-ketoester has been done at 0 ꢀC [10]. However, we
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.052

3846
J.M. Timonen et al. / European Journal of Medicinal Chemistry 46 (2011) 3845e3850
used a perchloric acid catalysed von Pechmann reaction (Scheme 1)
at room temperature. This was expected to diminish side-reactions
but may also have an effect on reaction time or yield. We conducted
every step, including the dissolving resorcinol to acid, the addition
The effects of the 7-hydroxycoumarins on NO production and
iNOS expression were investigated in macrophages activated by
exposing them to an inflammatory stimulus, lipopolysaccharide
(LPS), which activates the cells through a toll-like-receptor 4
(TLR4) pathway. To evaluate the mechanism of action, three of
the most effective compounds were selected for studies, in
which their effects on iNOS mRNA expression and on the acti-
of b-ketoester and reaction, at room temperature. The perchloric
acid is practical to use because it does not give rise to warming in
contrast to sulphuric acid, which heats up in pouring the reaction
mixture into the water. The desired coumarins were prepared from
vation of NF-kB, an important transcription factor for iNOS gene,
properly substituted
coumarin derivatives are presented in Scheme 1.
b
-ketoesters and resorcinols. The studied
were measured. In addition, effects of the compounds on the
expression of an inflammatory enzyme COX-2 (which produces
prostaglandins) and on a proinflammatory cytokine IL-6 were
also investigated. Measurements were carried out with two
Five of the twenty-two coumarins (14, 17, 18, 21 and 22)
reported here are new compounds. All the compounds were syn-
thesised by a modified method which has not previously been
reported in the literature. The differences are in principal used
catalytic acid and the conduction of the reaction at room temper-
ature. The mechanism of the von Pechmann reaction has been
presented in the literature. The mechanism points out the impor-
tance of an acidic catalyst by showing the ketoester activation step
in the beginning of the reaction [11].
Compounds 1 and 3 were prepared using a different method for
producing 4-unsubstituted compounds from resorcinol and ethyl-
propiolate. Dioxane was used as the solvent and zinc chloride as the
catalyst [12]. The reaction conditions had to be kept dry because of
the catalyst. This method gave lower yields than the acidic von
Pechmann reaction.
concentrations of the tested 7-hydroxycoumarins, 10
100 M.
mM and
m
The pharmacological studies on the newly synthesised
compounds were started by cytotoxicity testing. Cytotoxicity as
a contributing factor was ruled out by using a XTT test. The test
measures cells’ mitochondrial dehydrogenase activity that only
occurs in viable cells. Triton-X (0.1%) was used as a positive control
of cell death.
4. Results and discussion
4.1. Synthesis
Crude products were recrystallised from ethanol and to analyse
the purity and structure of the compounds we measured standard
1H NMR, 13C NMR and IR spectra. The purity of all compounds was
confirmed by combustion analysis. Also, the exact masses of the
[M ꢁ H]ꢁ ions of the studied compounds were measured. The
skeletal structure was studied by X-ray crystallographic determi-
nation of three compounds 10, 21 and 22.
Investigation of the crude products indicated good purity and in
consequence of room temperature the diminution of the side-
reactions was more significant than the elongated reaction time.
The monitoring of reactions by TLC indicated the reaction time to be
mostly from 4 to 6 h. Longer period did not increase the yield
remarkably.
The compound 15 differed from the others by both the needed
reaction time and the isolation method. The best result was ach-
ieved within a period of one and a half day. The precipitated
product started immediately to decompose after the reaction
mixture was poured into water. The extraction of diluted reaction
mixture with ethyl acetate, followed directly by neutralisation of
the organic layers with sodium bicarbonate solution was used to
protect the product. The combined organic phases were washed
several times with water before drying and evaporation.
The zinc chloride catalysed reaction used is very sensitive to
moisture and both dioxane and zinc chloride should be dried
carefully before the use. The perchloric acid catalysed von Pech-
mann reaction at elevated temperature to produce 4-unsubstituted
7-hydroxycoumarins has been reported in literature [5].
In the perchloric acid catalysed von Pechmann reaction used
starting materials were DL-malic acid and resorcinol. The study
indicated the acid being capable to activate malic acid partially but
at room temperature the reaction did not produce any detectable
coumarin structure.
3. Pharmacology
In the present study, we aimed to investigate the possible anti-
inflammatory effects of 7-hydroxycoumarins because they are
important metabolites of coumarins in man [8]. Since some
7-oxycoumarins have been reported as potential anti-inflammatory
compounds the study of 7-hydroxycoumarins may reveal new
possibilities to development of coumarin based drugs to treat
diseases caused by inflammation.
4.2. Inhibition of NO production
All of the compounds significantly inhibited LPS-induced NO
production when used at 100
mM concentrations, and nine
compounds inhibited it by more than 80% as compared to cells
treated with LPS alone. These nine compounds were 5-methyl (3),
3,4-dimethyl (6), 3,4,5-trimethyl (8), 3-ethyl-4-methyl (9), 3-ethyl-
4,8-dimethyl (13), 8-methoxy-4-propyl (18), 4-phenyl (19), 4-(-2-
FC6H4) (21) and 8-methoxy-4-phenyl (22) derivatives. Five of the
compounds (8, 18, 19, 21 and 22) also inhibited LPS-induced NO
production by more than 50% at the lower concentration tested
(10 mM) (Table 1). A highly selective iNOS inhibitor 1400W [13] was
used as a positive control compound. 1400W (1000
NO production in LPS-stimulated cells by 99%.
mM) inhibited
Scheme 1. Syntheses of 7-hydroxycoumarins via methods a and b.

J.M. Timonen et al. / European Journal of Medicinal Chemistry 46 (2011) 3845e3850
3847
Table 1
The three compounds inhibited LPS-induced iNOS mRNA expres-
sion by more than 50%. Activation of the transcription factor NF-
was investigated by measuring the nuclear translocation of the p65
subunit of NF- B by Western blotting. LPS induced NF- B trans-
location, and it peaked at 30 min after the addition of LPS. All three
tested 7-hydroxycoumarins inhibited LPS-stimulated NF- B p65
translocation by more than 65%. A previously known NF-
inhibitor PDTC [15] was used as a control compound, and when
used at 100 M concentrations it inhibited NF- B activation by
The effect of 7-hydroxycoumarins on LPS-induced NO production and iNOS protein
expression in J774 macrophages.
k
B
Compound
NO production
(inhibition %)
iNOS protein expression
(inhibition %)
k
k
10
mM
100
m
M
10
m
M
100 mM
k
kB
1
2
3
4
5
6
7
8
5.5 ꢂ 1.2
64.4 ꢂ 2.3
42.9 ꢂ 5.4
80.0 ꢂ 1.9
71.0 ꢂ 1.6
65.7 ꢂ 1.9
88.9 ꢂ 0.6
62.8 ꢂ 3.0
92.4 ꢂ 0.8
92.3 ꢂ 0.1
67.6 ꢂ 2.7
87.0 ꢂ 0.9
62.5 ꢂ 2.3
50.5 ꢂ 1.5
50.2 ꢂ 2.4
83.7 ꢂ 0.8
85.0 ꢂ 0.2
87.5 ꢂ 0.9
81.8 ꢂ 1.8
10.0 ꢂ 12.1
22.1 ꢂ 12.4
2.7 ꢂ 5.4
47.2 ꢂ 7.3
61.8 ꢂ 8.7
86.2 ꢂ 3.9
74.3 ꢂ 3.5
61.4 ꢂ 1.2
85.5 ꢂ 0.4
53.0 ꢂ 1.3
94.7 ꢂ 1.5
91.0 ꢂ 1.1
89.7 ꢂ 1.3
65.2 ꢂ 1.1
87.8 ꢂ 1.8
74.2 ꢂ 5.9
86.2 ꢂ 3.9
74.6 ꢂ 2.2
93.0 ꢂ 1.3
95.4 ꢂ 2.1
84.6 ꢂ 3.2
ꢁ14.2 ꢂ 2.5
9.2 ꢂ 2.7
m
k
ꢁ8.6 ꢂ 2.8
17.3 ꢂ 3.2
37.6 ꢂ 2.4
7.4 ꢂ 4.5
ꢁ19.7 ꢂ 8.6
20.1 ꢂ 7.0
28.6 ꢂ 6.7
46.5 ꢂ 7.5
49.6 ꢂ 1.6
7.5 ꢂ 6.4
more than 95%.
4.4. Inhibition of IL-6 production
51.8 ꢂ 3.1
43.3 ꢂ 1.4
25.3 ꢂ 1.3
46.5 ꢂ 1.9
38.5 ꢂ 2.0
21.7 ꢂ 6.2
15.8 ꢂ 1.5
57.0 ꢂ 2.0
54.8 ꢂ 2.9
50.4 ꢂ 3.3
71.5 ꢂ 3.6
9
We also tested the effects of 7-hydroxycoumarins on the LPS-
induced production of proinflammatory cytokine IL-6, by
measuring its concentration in the cell culture medium by immu-
noassay. IL-6 production in resting cells was very low, but it was
significantly enhanced when cells were activated by adding LPS.
10
13
14
16
17
18
19
21
22
25.3 ꢂ 4.2
18.9 ꢂ 3.2
7.8 ꢂ 4.3
18.0 ꢂ 8.3
2.7 ꢂ 5.4
13.4 ꢂ 6.7
46.5 ꢂ 5.6
48.8 ꢂ 6.0
55.9 ꢂ 2.3
With the higher concentration used (100 mM), eight of the studied
compounds (5, 6, 8, 9, 10,13, 19 and 21) inhibited IL-6 production by
more than 80%, and only two compounds (3 and 17) had an
inhibitory effect smaller than 30%. With the lower concentration
Mean ꢂ SEM, n ¼ 4.
used (10
for none of them was the inhibition over 30% (Table 2). Dexa-
methasone (10 M) was used as a control compound and inhibited
mM), ten compounds inhibited IL-6 production slightly but
m
In inflammation, NO is principally formed in a reaction cata-
lysed by iNOS which is an inducible enzyme. Therefore, we
investigated if the 7-hydroxycoumarins also effect on iNOS
expression, which was studied by Western blot analysis. Unsti-
mulated cells did not express detectable amounts of iNOS
protein, but LPS enhanced iNOS expression considerably. All
tested 7-hydroxycoumarins suppressed iNOS expression signifi-
IL-6 production by 74%.
4.5. Effect on COX-2 production
The effect of 7-hydroxycoumarins on LPS-stimulated COX-2
expression in J774 macrophages was also tested. None of the
studied eighteen compounds inhibited COX-2 expression in
cantly when studied at a 100 mM concentration, and ten of them
a significant manner (Table 3). Dexamethasone (10 mM) was used as
(3, 6, 8, 9, 10, 14, 17, 19, 21 and 22) inhibited iNOS protein
a control compound and inhibited COX-2 expression by 87%.
expression by more than 80%. With the lower concentration
(10
mM), most compounds had a minor inhibitory effect on
iNOS expression, and five of them (7, 8, 19, 21 and 22) inhibited
iNOS expression by more than 45% (Table 1). Recent study on
3-(10,10-dimethyl-allyl)-6-hydroxy-7-methoxycoumarin isolated
from Ruta graveolens L. indicated it to inhibit NO production and
4.6. Cytotoxicity
None of the compounds at concentrations of 100 mM showed
cytotoxic effects. XTT testing was carried out in similar conditions
where the experiments for anti-inflammatory activities were
carried out.
iNOS expression through suppression of NF-kB activation in
a macrophage cell line. In vivo experiments on endotoxin-
induced inflammation in mice the effect was 75% (i.p. injection,
1 mg/25 g body weight) [14]. Also, oxycoumarin derivatives have
been shown to inhibit iNOS expression and NO production, most
effective compounds were the 5,7-dimethoxycoumarins, which
Table 2
The effect of 7-hydroxycoumarins on LPS-induced IL-6 production in J774
macrophages.
inhibited NO production up to 95% with 50 mM in RAW245.7 cells
stimulated with LPS [7]. Sesquiterpene coumarins extracted from
roots of Ferula Fukanensis have also been shown to inhibit NO
production and iNOS expression (with IC50-values in range of
Compound
IL-6 production (inhibition %)
10
m
M
100 mM
1
2
3
4
5
6
7
8
3.2 ꢂ 3.5
4.6 ꢂ 5.2
34.1 ꢂ 1.1
44.1 ꢂ 2.5
4.2 ꢂ 4.9
9e25
Since NF-
expression we used pyrrolidinedithiocarbamate (PDTC), an inhib-
itor of NF- B activation [15] as a positive control for inhibition of
iNOS expression. PDTC (100 M) inhibited iNOS expression by 97%.
mM) [2].
k
B is an important transcription factor for iNOS
ꢁ22.5 ꢂ 2.6
14.5 ꢂ 4.8
17.5 ꢂ 1.9
16.7 ꢂ 1.5
21.2 ꢂ 4.4
ꢁ34.2 ꢂ 6.3
27.9 ꢂ 2.4
25.4 ꢂ 4.2
26.1 ꢂ 3.3
12.2 ꢂ 2.7
ꢁ0.9 ꢂ 7.0
10.7 ꢂ 5.2
11.0 ꢂ 4.1
ꢁ13.8 ꢂ 8.8
0.3 ꢂ 4.4
66.4 ꢂ 0.9
82.8 ꢂ 0.45
85.7 ꢂ 0.8
38.1 ꢂ 3.7
92.2 ꢂ 1.4
90.3 ꢂ 0.3
88.9 ꢂ 0.7
94.9 ꢂ 0.1
54.3 ꢂ 5.8
43.4 ꢂ 2.9
14.8 ꢂ 4.5
73.4 ꢂ 1.0
95.7 ꢂ 0.9
92.7 ꢂ 0.5
46.9 ꢂ 3.7
k
m
9
4.3. Anti-inflammatory mechanism of coumarins
10
13
14
16
17
18
19
21
22
To investigate the mechanisms of action in more detail,
we measured the effects of three of the most potent 7-hydroxycou-
marins (6, 8 and 9) on iNOS mRNA expression, and on the activation
of NF-kB. iNOS mRNA was measured by quantitative RT-PCR after
3
h
incubation with a
combination of LPS and the tested
ꢁ21.8 ꢂ 3.3
compounds. The iNOS mRNA levels were very low in resting cells,
but they increased significantly when the cells were exposed to LPS.
Mean ꢂ SEM, n ¼ 4.

3848
J.M. Timonen et al. / European Journal of Medicinal Chemistry 46 (2011) 3845e3850
Table 3
6. Experimental section
The effect of 7-hydroxycoumarins on LPS-induced COX-2 expression in J774
macrophages.
6.1. Materials and methods
Compound
COX-2 protein expression (inhibition %)
10 100 mM
The X-ray diffraction data were collected on a Nonius KappaCCD
m
M
diffractometer using Mo K
a
radiation (
l
¼ 0.710 73 Å). The crystals
1
2
3
4
5
6
7
8
ꢁ17.2 ꢂ 9.6
3.2 ꢂ 3.2
ꢁ20.4 ꢂ 8.9
22.7 ꢂ 6.7
ꢁ60.4 ꢂ 8.3
ꢁ17.5 ꢂ 8.6
7.8 ꢂ 3.2
were immersed in cryo-oil, mounted in a Nylon loop, and measured
at a temperature of 100 K. Chemicals were used as received if not
elsewise mentioned. Reactions were monitored by TLC using Merck
Kieselgel 60 F254 plates and ethyl acetateetoluene (1:1) as an
eluent. Melting points were measured with GWB Gallenkamp
Melting Point Apparatus and are uncorrected. NMR spectra were
measured either with a Bruker Avance 250 (250 MHz for 1H,
62.9 MHz for 13C) or with a Bruker Avance 400 FT NMR (400 MHz
for 1H, 100.6 MHz for 13C). IR spectra were measured with an Avatar
320 FT-IR. Combustion analyses were done using an Elementar
Analysensystem GmBH varioMICRO.
Reagents were obtained as following: goat polyclonal mouse
COX-2, rabbit polyclonal mouse iNOS, donkey anti-goat polyclonal,
and goat anti-rabbit polyclonal antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). All other reagents were
obtained from Sigma Chemical Co. (St. Louis, MO).
ꢁ44.1 ꢂ 9.2
ꢁ14.8 ꢂ 7.2
2.3 ꢂ 2.1
ꢁ57.7 ꢂ 9.3
ꢁ2.2 ꢂ 3.8
ꢁ31.7 ꢂ 6.5
ꢁ29.9 ꢂ 8.9
ꢁ11.1 ꢂ 8.3
ꢁ36.8 ꢂ 3.2
ꢁ6.8 ꢂ 11.6
ꢁ12.5 ꢂ 11.4
ꢁ15.4 ꢂ 4.2
ꢁ38.9 ꢂ 10.0
ꢁ47.1 ꢂ 8.3
ꢁ34.7 ꢂ 9.3
ꢁ59.1 ꢂ 8.4
3.7 ꢂ 6.8
ꢁ25.1 ꢂ 4.6
11.1 ꢂ 10.3
ꢁ73.2 ꢂ 13.0
25.0 ꢂ 2.7
21.0 ꢂ 1.5
ꢁ45.6 ꢂ 9.3
ꢁ16.3 ꢂ 11.9
ꢁ36.8 ꢂ 5.7
8.6 ꢂ 4.5
9
10
13
14
16
17
18
19
21
22
ꢁ0.4 ꢂ 6.6
ꢁ16.6 ꢂ 9.2
ꢁ34.7 ꢂ 5.3
Mean ꢂ SEM, n ¼ 4.
4.7. Structureeactivity relationship
6.1.1. Cell culture
J774 murine macrophages (American Type Culture Collection,
Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium
with glutamax-I. The culture media contained 10% heat-inactivated
When investigating the structureeactivity relationship, some
observations can be made. All three compounds containing phenyl
ring at position 4 were highly active in inhibiting NO production
and iNOS expression. From the five tested 3-alkyl substituted
compounds only 7-hydroxy-8-methoxy-3,4-dimethylcoumarin
(16) did not show high iNOS, NO and IL-6 inhibition activity. High
activities indicate the activation effect of 3-alkyl substitution to be
significant. The reason for lower activity of compound 16 is
supposed the methoxy group being capable to hinder the
membrane solubility. The effect is not clear, because in comparison
the 4-phenyl and the 4-phenyl-8-methoxy compounds (19 and 22)
the activity was only slightly lowered by methoxy group. The
reason for this difference could be based on the big electrostatic
effect of the phenyl which diminish the effect of methoxy group.
However, a lipophilic substituent at position 3 increases the activity
whereas the 3-fluorine does not activate the compounds.
foetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin,
and 250ng/mL amphotericinB (allfrom Gibco, Paisley, Scotland). Cells
were seeded on 24-well plates, and the cell monolayers were grown
for 72 h to confluence before the experiments were started and the
compounds of interest were added in fresh culture medium.
6.1.2. XTT test
Cell viability was tested by using Cell Proliferation Kit II, which
measures cells’ ability to metabolise sodium 30-[1-(phenyl-
aminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)
benzene sulphonic acid hydrate (XTT) to formazan by mitochon-
drial dehydrogenases that only occur in viable cells (Boehringer
Mannheim, Indianapolis, IN, USA).
6.1.3. Nitrite assays
5. Conclusions
At indicated time points, the culture medium was collected for
nitrite measurement, which was used as a measure of NO
Twenty 7-hydroxycoumarins were synthesised by
a
new
production. Culture medium (100 ml) was incubated with 100 ml of
modification of the perchloric acid catalysed von Pechmann reac-
tion and two by a zinc chloride catalysed reaction. The procedure
differed from earlier in the reaction temperature. Instead of clas-
sical heating the reaction was kept at room temperature. The
method is practical because it showed only a low appearance of
side-reactions and required only a little longer reaction time.
Also, 7-hydroxycoumarins inhibit iNOS expression and NO and
IL-6 production in a dose-dependent manner at non-cytotoxic
concentrations. Based on the experiments, the 7-hydroxycoumarins
also inhibit LPS-induced iNOS mRNA expression and nuclear trans-
Griess reagent (0.1% napthalethylenediamine dihydrochloride, 1%
sulfanilamine, and 2.5% H3PO4), and the absorbance was measured
at 540 nm. The concentration of nitrite was calculated with sodium
nitrite as the standard.
6.1.4. Preparation of cell lysates for Western blotting
At indicated time points, cells were rapidly washed with ice-
cold phosphate-buffered saline (PBS) that contained 2 mM sodiu-
morthovanadate. The cells were resuspended in a lysis buffer
containing 1% Triton-X, 50 mM NaCl, 10 mM Tris-base pH 7.4, 5 mM
EDTA, 0.5 mM phenylmethylsulfonylfluoride, 1 mM sodiumortho-
location (i.e. activation) of NF-
kB. The inhibition of COX-2 expression
is only slightly affected which increase their value as selective
inhibitors. Phenyl ring at position 4 or a lipophilic substituent at
position 3 increases the inhibition. However, the 3-fluorine does not
activate the compounds.
In conclusion, these findings suggest that 7-hydroxycoumarins
inhibit the early signalling mechanisms needed to induce tran-
scription of iNOS gene in inflammation and introduce them as
compounds with promising anti-inflammatory effects in conditions
complicated by increased iNOS expression and NO production.
vanadate, 40
sodiumpyrophosphate, and 10
After incubation for 15 min on ice, the lysates were centrifuged
(13 500 g, 5 min). The protein content of the supernatants was
measured by the Coomassie blue method.
m
M leupeptin, 50
m
g/mL aprotinin, 5 mM NaF, 2 mM
m
M N-octyl- -glucopyranoside.
b-D
6.1.5. Preparation of nuclear extracts for Western blotting
At indicated time points, the cells were rapidly washed with ice-
cold PBS and solubilised in hypotonic buffer A (10 mM HEPES-KOH

J.M. Timonen et al. / European Journal of Medicinal Chemistry 46 (2011) 3845e3850
3849
pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiotreitol, 0.2 mM
phenylmethylsulfonylfluoride, 10 g/mL leupeptin, 25 g/mL
A standard curve method was used to determine the relative
mRNA levels, as described in the Applied Biosystems User Bulletin:
a standard curve for each gene was created using RNA isolated from
LPS-stimulated J774 macrophages. Isolated RNA was reverse-
transcribed and the dilution series of cDNA ranging from 1 pg to
10 ng were subjected to real-time PCR. The obtained threshold
cycle values were plotted against the dilution factor to create
a standard curve. Relative mRNA levels in the test samples were
then calculated from the standard curve. When calculating the
results the iNOS mRNA levels were first normalised against GAPDH.
m
m
aprotinin, 0.1 mM EGTA, 1 mM sodiumorthovanadate, and 1 mM
NaF). After incubation for 10 min on ice, the cells were vortexed for
30 s and the nuclei were separated by centrifugation at 4 ꢀC,
21 000 g for 10 s. The pellet was resuspended in buffer C (20 mM
HEPES-KOH pH 7.9, 420 mM NaCl, 25% glycerol, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 mM dithiotreitol, 0.2 mM phenyl-
methylsulfonylfluoride, 10
mg/mL leupeptin, 25 mg/mL aprotinin,
0.1 mM EGTA, 1 mM sodiumorthovanadate, and 1 mM NaF) and
incubated on ice for 20 min. Nuclei were vortexed for 30 s and
nuclear extracts were obtained by centrifugation at 4 ꢀC, 21 000 g
for 2 min. The protein content of the supernatant was measured by
the Coomassie blue method. The samples were boiled in a SDS
sample buffer and stored at ꢁ20 ꢀC.
6.1.8. Enzyme-linked immunosorbent assay (ELISA)
Culture medium samples were kept at ꢁ20 ꢀC until assayed. The
concentration of mouse IL-6 (DuoSetÒ ELISA, R&D Systems Europe
Ltd, Abindgon, U.K.) was determined by ELISA according to the
manufacturer’s instructions.
6.1.6. Western blot analysis
After boiling for 5 min, equal aliquots of protein (20
mg) were
6.1.9. Statistics
loaded onto a 10% SDS-polyacrylamide electrophoresis gel and
electrophoresed for 4 h at 100 V in a buffer containing 95 mM
TriseHCl, 960 mM glycine, and 0.5% SDS. After electrophoresis,
the proteins were transferred to a Hybond enhanced chem-
iluminescence nitrocellulose membrane (Amersham, Buck-
inghamshire, U.K.) with a semi-dry blotter at 2.5 mA/cm2 for 60 min.
After transfer, the membrane was blocked inTBS/T (20 mM Tris-base
pH 7.6, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat milk for
The results are expressed as the mean ꢂ S.E.M. Statistical
significances of the differences were calculated by analyses of
variance, supported by the Dunnett’s multiple comparisons test.
Differences were considered significant at P < 0.05.
6.2. Preparation of studied compounds
6.2.1. General method for 4-unsubstituted 7-hydroxycoumarins (1, 3)
Properly substituted resorcinol (100 mmol) was dissolved in
dioxane (100 ml) and ethylpropiolate (100 mmol), and dried zinc
chloride (100 mmol) was added. The mixture was refluxed for
24 h. After cooling to room temperature, 100 ml of 5% HCl was
added slowly. After addition, the mixture was concentrated to half
volume and kept at þ4 ꢀC. The precipitate was filtered and dried at
room temperature. The product was recrystallised from
ethanol. Analytical data of compounds 1 and 3 is presented in
Supplementary data.
1 h at room temperature and incubated overnight at 4 ꢀC with NF-
kB
p65, COX-2 or iNOS antibodies in TBS/T containing 5% nonfat milk.
Thereafter, the membrane was washed 4 times in TBS/T for 5 min,
incubated with a secondary antibody coupled to horseradish
peroxidase in the blocking solution for 0.5 h at room temperature,
and washed four times with TBS/T for 5 min. A bound antibody was
detected using a SuperSignal West Pico chemiluminescent substrate
(Pierce, Cheshire, U.K.) and a FluorChem 8800 imaging system
(Alpha Innotech Corp., San Leandro, CA). The chemiluminescent
signal was quantified by using FluorChem software version 3.1.
6.2.2. General method for 4-substituted 7-hydroxycoumarins
(2, 4e22)
6.1.7. RNA extraction and real-time RT-PCR
At the indicated time points, cell monolayers were rapidly
washed with ice-cold PBS, and cells were homogenised using
a QIAshredder (QIAGEN, Valencia, CA). RNA extraction was carried
out with the use of an RNeasy kit for isolation of total RNA (QIA-
GEN). Total RNA (25 ng) was reverse-transcribed to cDNA using
TaqMan Reverse Transcription reagents and random hexamers
(Applied Biosystems, Foster City, CA). cDNA obtained from the RT
reaction (amount corresponding to approximately 1 ng of the total
RNA) was subjected to PCR using a TaqMan Universal PCR Master
Mix and an ABI PRISM 7000 Sequence detection system (Applied
Biosystems). The primer and probe sequences and concentrations
were optimised according to the manufacturer’s guidelines in the
TaqMan Universal PCR Master Mix Protocol part number 4304449
revision C, and were: 50-CCTGGTACGGGCATTGCT-30, 50-GCTCATGC
GGCCTCCTT-30 (forward and reverse mouse iNOS primers, respec-
tively, both 300 nM), 50-CAGCAGCGGCTCCATGACTCCC-30 (mouse
iNOS probe of 150 nM, containing 6-FAM as 50-reporter dye and
TAMRA as 30-quencher), 50-GCATGGCCTTCCGTGTTC-30 (mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward
primer, 300 nM), 50-GATGTCATCATACTTGGCAGGTTT-30 (mouse
GAPDH reverse primer, 300 nM), and 50-TCGTGGATCT-
GACGTGCCGCC-30 (mouse GAPDH probe, 150 nM, containing
6-FAM as 50-reporter dye and TAMRA as 30-quencher). The PCR
reaction parameters were as follows: incubation at 50 ꢀC for 2 min,
incubation at 95 ꢀC for 10 min, and, thereafter, 40 cycles ofdena-
turation at 95 ꢀC for 15 s and annealing and extension at 60 ꢀC for
1 min. Each sample was determinated in duplicate.
30 mmol of properly substituted resorcinol was dissolved in 30 ml
of perchloric acid. 30 mmol of b-ketoester was added and the reac-
tion mixture was stirred at room temperature until TLC showed that
resorcinol had reacted. The mixture was poured into 250 ml of cold
water and the precipitate was filtered and dried at room temperature
and recrystallised from ethanol. Analytical data of compounds 2e13,
15, 16, 19 and 20 is presented in Supplementary data.
6.2.3. 7-Hydroxy-5-methyl-4-propyl-2H-benzo-1-pyran-2-one (14)
Yield 86%. mp 165e167 ꢀC. TLC Rf ¼ 0.685 (ethyl acetate/toluene,
1:1, v/v). 1H NMR (d6-DMSO)
d
(ppm): 0.96 (t, 3H, J ¼ 7.5 Hz, 30-CH3),
1.61 (sxt, 2H, J ¼ 7.4 Hz, 20-CH2), 2.28 (s, 3H, 5- CH3), 2.90 (t, 2-H,
J ¼ 7.9 Hz,10-CH2), 6.02 (s,1H, H-3), 6.59 (d,1H, J ¼ 1.47 Hz, H-6), 6.63
(d, 1H, J ¼ 1.47 Hz, H-8), 10.61 (s, 1H, OH). 13C NMR (d6-DMSO)
d
(ppm): 13.71, 20.96, 22.38, 37.17, 105.74, 107.84, 111.25, 112.06,
142.52, 155.10, 155.86, 158.00, 159.83. IR (KBr): 3231, 2962, 2875,
1681,1628,1601,1512,1457,1398,1334,1283,1250,1213,1153,1095,
1047,1015, 996, 926, 887, 825, 705. Anal. calcd for C13H14O3: C 71.54,
H 6.47; found C 71.37, H 6.59. ESI-MS (m/z) 217.0876 ([M ꢁ H]ꢁ).
6.2.4. 3-Fluoro-7-hydroxy-8-methoxy-4-methyl-2H-benzo-1-
pyran-2-one (17)
Yield 96%. mp 168e170 ꢀC. TLC Rf ¼ 0.625 (ethyl acetate/toluene,
1:1, v/v). 1H NMR (d6-DMSO)
d
(ppm): 2.32 (d, 3H, J ¼ 2.9 Hz, 4-
CH3), 3.83 (s, 3H, OCH3), 6.95 (d, 1H, J ¼ 8.9 Hz, H-6), 7.34 (d, 1H,
J ¼ 8.9 Hz, H-5), 10.37 (br, 1H, OH). 13C NMR (d6-DMSO)
d (ppm):
10.19, 61.77, 113.94, 115.21, 121.27, 133.95, 134.16, 136.11, 146.04,

3850
J.M. Timonen et al. / European Journal of Medicinal Chemistry 46 (2011) 3845e3850
154.29, 157.07. IR (KBr): 3259, 3006, 2943, 2842, 1716, 1641, 1600,
1514, 1464, 1437, 1384, 1361, 1329, 1291, 1242, 1198, 1160, 1115, 1021,
1000, 936, 822, 749. Anal. calcd for C11H9O4F: C 58.93, H 4.05; found
C 58.73, H 4.40. ESI-MS (m/z) 223.0411 ([M ꢁ H]ꢁ).
acknowledged. Professor Gregory Watson is highly appreciated for
revising of language. The excellent technical assistance of Ms Meiju
Kukkonen and the skillful secretarial help of Mrs Heli Määttä are
gratefully acknowledged.
6.2.5. 7-Hydroxy-8-methoxy-4-propyl-2H-benzo-1-pyran-2-one (18)
Yield 89%. mp 129e131 ꢀC. TLC Rf ¼ 0.603 (ethyl acetate/toluene,
Appendix. Supplementary information
1:1, v/v). 1H NMR (d6-DMSO)
d
(ppm): 0.92 (t, 3H, J ¼ 7.4 Hz, 30-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.05.052.
CH3), 1.56 (sxt, 2H, J ¼ 7.4 Hz, 20-CH2), 2.66 (t, 2H J ¼ 7.4 Hz, 10-CH2),
3.77 (s, 3H, OCH3), 6.07 (s,1H, H-3), 6.84 (d,1H, J ¼ 8.8 Hz, H-6), 7.36
(d, 1H, J ¼ 8.8 Hz, H-5), 10.48 (br, 1H, OH). 13C NMR (d6-DMSO)
References
d
(ppm): 13.58, 21.37, 32.90, 60.54, 109.28, 112.06, 113.04, 120.19,
134.28, 147.85, 153.42, 157.41, 160.15. IR (KBr): 3268, 2963, 2938,
2873,1726,1680,1601,1512,1457,1434,1387,1352,1264,1201,1166,
1056, 1006, 933, 843, 809, 721. Anal. calcd for C13H14O4: C 66.66, H
6.02; found C 66.54, H 6.15. ESI-MS (m/z) 233.0819 ([M ꢁ H]ꢁ).
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6.2.6. 7-Hydroxy-4-(2-fluoro-phenyl)-2H-benzo-1-pyran-2-one (21)
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d
(ppm): 104.43, 112.46, 113.91, 115.27, 117.77, 124.42, 126.98, 129.77,
132.61, 133.73, 152.10, 157.01, 158.40, 161.81, 163.47. IR (KBr): 3344,
3155,1693,1597,1563,1490,1446,1377,1308,1263,1237,1152,1119,
1090, 1001, 943, 849, 817, 765. Anal. calcd for C15H9O3F: C 70.31, H
3.54; found C 70.13, H 3.85. ESI-MS (m/z) 255.0463 ([M ꢁ H]ꢁ).
6.2.7. 7-Hydroxy-8-methoxy-4-phenyl-2H-benzo-1-pyran-2-one (22)
Yield 94%. mp 181 ꢀC. TLC Rf ¼ 0.622 (ethyl acetate/toluene,1:1, v/
v). 1H NMR (d6-DMSO)
d (ppm): 3.86 (s, 3H, OCH3), 6.18 (s, 1H, H-3),
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6.86 (d, 1H, J ¼ 8.7 Hz, H-6), 7.02 (d, 1H, J ¼ 8.7 Hz, H-5), 7.55 (m, 5H,
C6H5), 10.54 (br, 1H, OH). 13C NMR (d6-DMSO)
d
(ppm): 60.53, 110.37,
111.50, 113.20, 121.99, 128.35, 128.72, 129.50, 134.54, 135.12, 148.28,
153.82, 155.66, 159.70. IR (KBr): 3344, 1695, 1602, 1564, 1474, 1429,
1362,1308,1287,1201,1119,1078, 849, 823. Anal. calcd for C16H12O4: C
71.64, H 4.51;foundC 71.27, H4.76. ESI-MS(m/z) 267.0660([Mꢁ H]ꢁ).
Acknowledgements
Financial support from the National Graduate School of Organic
Chemistry and Chemical Biology, University of Joensuu, University
of Eastern-Finland, The Academy of Finland and the Competitive
Research Funding of the Tampere University Hospital is gratefully
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