T. Symeonidis et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1139–1142
1141
Table 2
activity. No differences were observed between the hydrox-
ycoumarins and the hydroxybenzocoumarins.
Inhibition of lipid peroxidation at 100
activity (O2 %) at 100
l
M (LP %); superoxide radical scavenging
ꢁÅ
l
M; in vitro inhibition of soybean lipoxygenase (LO) % at
Compounds 3a,b,f,g and 3i were tested for their anti-inflamma-
tory activity in vivo (dose ip 0.01 mmol/kg body weight). The
in vivo anti-inflammatory effects of the tested coumarins were
assessed by using the functional model of carrageenin-induced
rat paw edema (CPE)22 and are presented in Table 2, as percent
inhibition of induced rat paw edema. After 3.5 h, compounds 3b,
3f and 3g induced very low protection (15–18%) against carra-
geenin-induced paw edema while the reference drug indometha-
cin (IMA) induced 47% protection at an equivalent dose.
Compound 3a did not inhibit carrageenin-induced rat paw edema
whereas compound 3i (a hydroxyl benzocoumarin) presents the
higher inhibition among the tested compounds (39%). The side of
ring’s condensation seems to influence the biological response
[f] < [h] (compounds 3f, 3g < 3i).
In conclusion, the broad spectrum of the observed antioxidant
activity of the majority of the examined coumarins allows us to
propose them as templates in the design of compounds useful in
treating human diseases that involves reactive oxygen species
(ROS). Their synthesis is almost simple with moderate to high
yields. Most of them are potent superoxide anion scavengers and
inhibit in vitro lipid peroxidation. Antioxidant power might be
important in the inhibition of lipid peroxidation. Compound 3i pre-
senting higher LO inhibitory activity among the tested hydrox-
ybenzocoumarins, was found to present a promising antioxidant
profile and 39% inhibition on carrageenin-induced rat paw edema.
100 lM; inhibition % of induced carrageenin rat paw edema (CPE %) at 0.01 mmol/kg
body weight
ꢁÅ
No
LP % at 100
l
M
O2 % at 100
l
M
LO % at 100
lM
CPE %a
3a
3b
3c
3d
3e
3f
3g
3h
83
28
24
30
No
57
100
42
100
nt
65
56
54
61
60
No
79
68
61
nt
IC50 = 43
37%
3%
27%
25%
38%
No
12%
IC50 = 100
15%
l
M
No
18*
15*
18*
3i
l
M
M
39*
30*
55*
Coumarin
7-CH3-coumarin nt
CA
nt
71
89%
IC50 = 600
l
Trolox
IMA
73
47*
CA, caffeic acid; IMA, indomethacin; No, no result under the experimental condi-
tions; each experiment was performed at least in triplicate and the standard
deviation of absorbance was less than 10% of the mean.
a
Statistical studies were done with student’s t-test.
*
p < 0.01.
observed between the hydroxycoumarins and the hydroxy-
benzocoumarins.
It is consistent thatrates of reactiveoxygen species (ROS) produc-
tion are increased in most diseases.32 Cytotoxicity of O2 and H2O2
ꢁÅ
Acknowledgments
in living organisms is mainly due to their transformation into ÅOH,
reactive radical metal complexes and 1O2. During the inflammatory
process, phagocytes generate the superoxide anion radical at the in-
flamed site. Enzymatic superoxide anion radicals were generated by
a hypoxanthine and xanthine oxidase (XOD) reaction system.33 At
pH 7.4 superoxide anion reduces the tetrazolium blue into formasan
blue (kmax = 560 nm). The production of superoxide was estimated
by the nitroblue tetrazolium method. The majority of the com-
The authors gratefully acknowledge support for part of this
work through the program PYTHAGORAS II of EPEAEK II (MIS:
97436/073). We would like to thank Dr. C. Hansch and Biobyte
Corp. 201 West 4th Street, Suite 204, Claremont, CA 91711, USA
for free access to the C-QSAR program.
pounds present high scavenging activity at 100 lM (54–79%) com-
References and notes
pared to caffeic acid used as a standard (71%) (Table 2), with the
exception of compound 3f which does not show any activity.
Compounds were further evaluated for inhibition of soybean
lipoxygenase LO by the UV absorbance based enzyme assay.22 Lip-
oxygenases oxidize certain fatty acids at specific positions to
hydroperoxides that are the precursors of leukotrienes, which con-
tain a conjugated triene structure. It is known that soybean lipoxy-
genase, which converts linoleic to 13-hydroperoxylinoleic acid, is
inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs)34,35
in a qualitatively similar way to that of the rat mast cell lipoxyge-
nase and may be used in a reliable screen35 for such activity. For
compounds 3a and 3i the IC50 values were determined. Compound
3a is the most potent derivative. The most of the LO inhibitors are
antioxidants or free radical scavengers,36 since lipoxygenation
occurs via a carbon-centered radical. Some studies suggest a rela-
tionship between LO inhibition and the ability of the inhibitors
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.
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