Synthesis and evaluation of the antioxidant and anti-inflammatory activity of novel coumarin-3-aminoamides and their alpha-lipoic acid adducts

Article abstract of DOI:10.1016/j.ejmech.2008.12.027

In the present work a series of novel coumarin-3-carboxamides and their hybrids with the alpha-lipoic acid were designed, synthesized and tested as potent antioxidant and anti-inflammatory agents. The new compounds were evaluated for their antioxidant act

Full text of DOI:10.1016/j.ejmech.2008.12.027

European Journal of Medicinal Chemistry 44 (2009) 3020–3026
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of the antioxidant and anti-inflammatory
activity of novel coumarin-3-aminoamides and their alpha-lipoic
acid adducts
,
a
Georgia Melagraki a, Antreas Afantitis a, Olga Igglessi-Markopoulou a , Anastasia Detsi ,
*
Maria Koufaki b, Christos Kontogiorgis c, Dimitra J. Hadjipavlou-Litina c
,
*
a Laboratory of Organic Chemistry, School of Chemical Engineering, National Technical University of Athens, 9, Heroon Polytechniou Street, Zorgafou Campus, Athens 157 73, Greece
b Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48, Vas. Constantinou Avenue, Athens 116 35, Greece
c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2008
Received in revised form
26 November 2008
Accepted 30 December 2008
Available online 19 January 2009
In the present work a series of novel coumarin-3-carboxamides and their hybrids with the alpha-lipoic
acid were designed, synthesized and tested as potent antioxidant and anti-inflammatory agents. The new
compounds were evaluated for their antioxidant activity, their activity to inhibit in vitro lipoxygenase
and their in vivo anti-inflammatory activity. In general, the derivatives were generally found to present
antioxidant and anti-inflammatory activities. Discussion is made based on the results for the structure–
activity relationships in order to define the structural features required for activity.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Coumarins
Lipoic acid
Antioxidant
Anti-inflammatory
1. Introduction
anticoagulant [9], antibacterial [10,11], anticancer [12], anthel-
minthic [13], anti-inflammatory [14–16] and antioxidant activities
The association of antioxidants with inflammation stems from
the recognition that free radicals are produced during the inflam-
matory process by macrophages. It has been reported that Reactive
Oxygen Species (ROS) are involved in the cyclooxygenase- and
lipoxygenase-mediated conversion of arachidonic acid into proin-
flammatory intermediates. On this basis, several natural and
synthetic antioxidants have been tested and shown to possess anti-
inflammatory properties [1,2].
Coumarins form an elite class of compounds, which occupy
a special role in nature. Pharmacologically, coumarins are flavo-
noids along with a range of other compounds. Coumarins and their
derivatives have been found to exhibit a variety of biological and
pharmacological activities and have raised considerable interest
because of their potential beneficial effects on human health [3,4].
They have been reported to possess among others: anti-HIV [5–8],
[17–19]. As a result, coumarins and their derivatives have been the
subject of extensive investigations.
The coumarin nucleus incorporates the styryl carbonyl moiety
into a rigid framework. It has been reported that styryl carbonyl
derivatives possess appreciable anti-inflammatory activity [20–21].
Moreover, coumarin and related derivatives have been used as
inhibitors of lipoxygenase (LOX) and cyclooxygenase (COX) path-
ways of arachidonic acid metabolism [22]. One of the most well-
studied coumarin-based anti-inflammatory drugs is cloricromene
(8-monochloro-3-b-diethylaminoethyl-4-methyl-7-ethoxy-carbonyl-
methoxy coumarin), and several synthetic analogues of this
compound have been recently reported and tested for their anti-
inflammatory and antioxidant activities [23].
In addition, many coumarin derivatives have special ability to
scavenge ROS and to influence processes involving free radical-
injury [24,25].
The 4-hydroxycoumarin moiety, widely spread among
coumarin natural products, has been the molecular template for
the synthesis of a variety of analogues exhibiting important bio-
logical activity. Characteristic examples include warfarin,
* Corresponding authors.
E-mail addresses: ojmark@chemeng.ntua.gr (O. Igglessi-Markopoulou), hadji-
pav@pharm.auth.gr (D.J. Hadjipavlou-Litina).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.027

G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
3021
Our work reveals that some of the novel hybrid compounds are
potent leads and may be useful in the prevention of human diseases
attributed to free radical damage.
O
OH
O
OH
O
2. Chemistry
O
O
The synthesis of the target compounds includes three steps
briefly described as follows: i) Synthesis of functionalized 3-
ethoxycarbonyl 4-hydroxycoumarins, ii) synthesis of aliphatic and
aromatic coumarin-3-carboxamides, using aliphatic and aromatic
diamines and iii) reaction of the resulting aminoamides with acti-
vated lipoic acid. The above steps are shown in Schemes 1–3.
Based on our previous work, an efficient short-step synthesis
was used to produce functionalized 3-ethoxycarbonyl 4-hydrox-
ycoumarins [39]. These compounds were synthesized via a C-
acylation–cyclization reaction of diethyl malonate with the N-
hydroxybenzotriazolyl esters of functionalized acetyl salicylic acids.
Our strategy involves activation of the functionalized acetyl sali-
cylic acids 1, 2 using N-hydroxybenzotriazole (HOBt) and N,N-
dicyclohexylcarbodiimide (DCC) and condensation of the resulting
N-hydroxybenzotriazolyl esters with the anion of diethyl malonate.
The intermediates 3, 4 were converted to the corresponding 3-
ethoxycarbonyl 4-hydroxycoumarins 5, 6 via an intramolecular
condensation reaction, using methanolic 10% hydrochloric acid
solution (Scheme 1).
Reaction of coumarins 5, 6 with the appropriate aliphatic
diamines (1,2-ethylenediamine, 1,6-hexamethylenediamine and
1,8-octamethylenediamine) and 1,2-phenylenediamine in refluxing
toluene yielded the desired coumarin-3-aminocarboxamides 7–13
(Scheme 2), as solids which precipitated in the reaction solution.
The compounds were isolated by filtration and washing with
diethylether, providing analytically pure compounds without the
need of further purification.
phenprocoumon
warfarin
OH
H
OCONH2
OH
MeO
N
O
O
O
O
O
novobiocin
Fig. 1.
a synthetic compound used as a rodenticide and anticoagulant and
phenprocoumon with antiviral and anti-HIV activities (Fig. 1) [3]. In
addition, aminocoumarin analogues such as novobiocin, chloro-
biocin, coumermycin and simocyclinone, which contain an amide
bond at position 3 of the heterocyclic ring, are potent antibiotics
[26,27]. Moreover, novel 4-hydroxycoumarin-3-carboxamide
derivatives have been synthesized as potential drugs for the
treatment of Insulin Dependent Diabetes Mellitus (IDDM) [28].
The potential of 4-hydroxycoumarin derivatives as anti-
inflammatory and antioxidant agents, prompted us to design and
synthesize a series of novel coumarin analogues bearing the 3-
carboxamide functionality. Analogous carboxamides based on the
structure of 4-hydroxy-quinolinone have been recently synthesized
by our group and led to compounds with combined antioxidant and
anti-inflammatory activities [29].
Activation of lipoic acid was accomplished by treatment of
racemic alpha-lipoic acid with N-hydroxysuccinimide and DCC in
dichloromethane. The resulting ester 14 is easily prepared in high
yields, can be stored for a long time and the N-hydroxysuccinimide
formed subsequently as a byproduct is a water-soluble compound
and can be efficiently removed from the reaction mixture.
The synthesis of hybrid compounds 15–19 was achieved by
dissolving equimolar amounts of compounds 7–13 with the active
ester 14 in dichloromethane containing a few drops of dime-
thylformamide in order to facilitate dissolution. The desired prod-
ucts were obtained after stirring for 48 h, at room temperature, in
the absence of light, followed by aqueous work-up and flash
column chromatography (Scheme 3).
Lipoic acid (1,2-dithiolane-3-valeric acid, LA) is a naturally
occurring compound present in all kinds of prokaryotic and
eukaryotic cells and synthesized by animals and humans. LA exists
as both the reduced dithiol form (dihydrolipoic acid – DHLA) and
the oxidized disulfide form [30,31]. A great deal of attention has
been paid to the antioxidant activity of the LA/DHLA redox couple
and their roles as biological thiol antioxidants since they have been
shown to possess remarkable antioxidant activity in vitro and in
vivo and have been described as universal antioxidants effectively
reacting with ROꢀ2, ascorbyl radicals, HOꢀ, NOꢀ, tocopheryl radicals,
Oꢀ2ꢁ and hypochlorous acid [32–34].
The combination of the distinct pharmacophores of two
different biologically active compounds in the same structure, has
been previously reported in literature and is highly likely to lead to
hybrid compounds with significant activity [35–38]. Recently we
have published some novel quinolinone-3-aminoamides bearing
a methyl or phenyl group on the nitrogen heteroatom incorporating
the alpha-lipoic acid (LA) moiety and we have examined the
influence of this modification on the anti-inflammatory/antioxi-
dant activity of the aminoamides [29]. Therefore, we decided to
additionally incorporate alpha-lipoic acid to the coumarin scaffold.
3. Biology
In acute toxicity experiments, the in vivo examined compounds
did not present toxic effects in doses up to 0.5 mmol/kg body
weight. In almost all cases the animals were survived (80–90%) and
looked normal both macroscopically and by autopsy. These results
indicate that the synthesized compound demonstrates low general
toxicity.
OH
R
COCH(CO2Et)2
OCOCH3
(iii)
R
COOH
(i), (ii)
R
CO2Et
OCOCH3
O
O
1 R = H
2 R = CH3
3 R = H
4 R = CH3
5
6
R = H
R = CH3
Scheme 1. Synthesis of 3-ethoxycarbonyl 4-hydroxycoumarins. Reagents and conditions: (i) HOBt, DCC, THF; (ii) NaH, diethyl malonate, THF; (iii) HCl/MeOH 10%.

3022
G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
OH
O
OH
4
O
9
potent. Their potency remains high even in lower doses (Table 1).
The nature and the presence of moiety X within the coumarin-3-
carboxamides seem to influence the inhibition values in compar-
ison to the starting materials 5 and 6. Thus, amides 7–10 derived
from compound 5 present a decrease in the induced carrageenin rat
paw edema (CPE) % values. Between compounds 7–9 the increase of
the number of the CH2 groups (from 2 to 8) led to a decrease in
activity. The decrease in the presence of an ortho-phenyl-diamine is
lower (compound 10 ¼ 57%, compound 5 ¼ 63%, Table 1). The
amides 11–13, derived from compound 6 led to more potent
derivatives. However the observed differences in CPE % values
among 11–13 are not significant.
5
6
4a
8a
3
R
CO2Et
R
(i)
N
H
X
NH2
2
7
O
O
O
8
5 R = H
6 R = CH3
R
H
H
H
X
7
(CH2)2
(CH2)6
(CH2)8
8
9
10
H
Lipoic acid–coumarin-3-aminoamides hybrids showed higher
anti-inflammatory activity than lipoic acid alone (29.6% inhibition
in the carrageenin-induced rat paw edema). The presence of the
lipoic group in the adducts 16 and 19 is correlated with higher
activity in comparison to compounds 8 and 12. However the adduct
17 presents lower activity than compound 9 (a coumarin-3-car-
boxamide derivative). The results show that, in the case of the
synthesized coumarin derivatives, overall lipophilicity does not
increase in parallel to inhibition (Table 1), with the exception of
compounds 12 and 19 (compound 12 ¼ c log P 4.01, CPE % ¼ 59 and
compound 19 ¼ c log P 6.18. and CPE % ¼ 73).
11
12
CH3
CH3
(CH2)2
(CH2)8
13
CH3
Scheme 2. Synthesis of coumarin-3-carboxamides. Reagents and conditions: (i)
diamine, toluene, 110 ꢂC.
3.1. In vivo anti-inflammatory activity
The in vivo anti-inflammatory effects of the tested coumarins
were assessed by using the functional model of carrageenin-
induced rat paw edema and are presented in Table 1, as percent
inhibition of induced rat paw edema. Acute inflammation is due to
the release of chemical mediators, which cause edema as a result of
extravasations of fluid and proteins from the local microvasculature
and accumulation of polymorphonuclear leukocytes at the
inflammatory site. Carrageenin-induced inflammation is a non-
specific inflammation resulting from a complex of diverse media-
tors [40]. This model is conventional, sensitive, and accepted for
screening of newer anti-inflammatory agents [41]. Further, this
model reliably predicts the anti-inflammatory efficacy based on the
inhibition of prostaglandin amplification [42]. As shown in Table 1,
all the investigated compounds inhibited carrageenin-induced paw
edema. The protection ranged from 41 to 73% while the reference
drug, indomethacin, induced 47% protection at an equivalent dose.
The coumarin–lipoic acid conjugates 16 and 19 were found highly
3.2. In vitro antioxidant activity
It is well known that free radicals play an important role in the
inflammatory process [43]. Many non-steroidal anti-inflammatory
drugs have been reported to act either as inhibitors of free radical
production or as radical scavengers [44]. Consequently, compounds
with antioxidant properties could be expected to offer protection in
rheumatoid arthritis and inflammation and to lead to potentially
effective drugs. Thus, we tested the novel amides and hybrids with
regard to their antioxidant ability and in comparison to well-known
antioxidant agents e.g. nordihydroguaiaretic acid and trolox (Tables
1 and 2). The potential of the antioxidant activity is shown in Tables
1 and 2. The antioxidant activity has been evaluated in two in vitro
Table 1
Competition % of compounds with DMSO for hydroxyl radical (HOꢀ %); inhibition of
soybean lipoxygenase percent LOX %; percent inhibition of induced carrageenin rat
paw edema (CPE %) at 0.01 mmol/kg body weight. Calculated lipophilicity c log P51
.
Compounds
c log P [51]
ꢀOH %a
LOX %b
CPE %c 0.01 mmol/kg
OH
O
O
O
O
5
6
7
8
2.83
3.33
1.21
2.46
3.51
2.26
1.71
4.01
2.76
4.62
91
81
91
100
no
93
93
100
52
no
23
14
30
22
18
63**
41*
59**
49**
48**
57*
59**
59**
60**
72d **
R
N
H
X
NH2
N
O
+
S
O
7-13
S
O
9
14
10
11
12
13
16
no
100
100
100
96
(i)
O
OH
O
61e
43*
73d
60e
*
X
R
N
H
N
H
17
19
5.68
6.18
no
100
33
8.3
*
*
S
S
O
O
R
H
Indomethacin
Lipoic acid
NDGA
nt
38.6
nt
nt
47**
29.6**
nt
X
2.39
29.1
84
nt
(CH2)2
(CH2)6
(CH2)8
(CH2)2
(CH2)8
15
16
17
H
Trolox
88.2
nt
H
**P < 0.01, *P < 0.05; no: no action under the reported experimental conditions;
nt: not tested; Nordihydroguaiaretic acid (NDGA).
CH3
CH3
18
19
a
Final concentration for the tested compounds 0.1 mM.
Final concentration for the tested compounds 0.1 mM.
Statistical studies were done with student’s t-test.
The in vitro results were performed at 0.01 mmol/kg.
b
c
d
Scheme 3. Synthesis of coumarin–lipoic acid conjugates. Reagents and conditions: (i)
CH2Cl2, DMF, r.t.
e
The in vitro results were performed at 0.001 mmol/kg.

G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
3023
Table 2
radicals are considered to be responsible for some of the tissue
damages occurring in inflammation. Hydroxy radicals formed in
the body, can lead to the generation of carbon-centered and peroxyl
radicals. It has been claimed that hydroxyl radical scavengers could
serve as protectors, thus increasing prostaglandin synthesis.
The competition of coumarins with DMSO for HOꢀ generated by
the Fe3þ/ascorbic acid system, expressed as percent inhibition of
formaldehyde production, was used for the evaluation of their
hydroxyl radical scavenging activity. In these experiments (Table 1)
compound 9 and its adduct compound 17 did not show any inhi-
bition at 0.1 mM. Compound 10 also did not present any activity.
Compounds 8, 11–13, 19 and 16 were found to be very potent fol-
lowed by compounds 5 and 7 (equipotent) and 6. It is important
that compounds 8 and 12 and their adducts 16 and 19 are highly
potent at 0.1 mM. In compound 9 with a (CH2)8 methylenic chain
the competition disappears. The ‘‘mother compound’’ coumarin
presents 78% competition at the concentration of 0.1 mM showing
that the coumarin nucleus implicated by itself in the scavenging
procedure.
Interaction with DPPH-reducing activity % (RA %).
Compounds
0.1 mM
0.1 mM
0.5 mM
0.5 mM
RA % 20 min
RA % 60 min
RA % 20 min
RA % 60 min
5
6
7
8
9
10
11
12
13
16
17
19
15
18
42
3
27
8
25
3
20
20
No
5
13
33
No
5
19
83
65
78
78
12
25
87
7
17
90
48
58
74
15
21
69
20
81
8
9
93
59
68
65
18
21
65
27
83
85
70
72
70
23
32
89
5
Lipoic acid
NDGA
97
98
No: no action under the reported experimental conditions; Nordihydroguaiaretic
acid (NDGA); the final concentrations of compounds were 0.1 mM and 0.5 mM.
The assay for lipoxygenase (LOX) activity was carried out
according to the UV absorbance based enzyme assay [47] using
soybean lipoxygenase. While one may not extrapolate the quanti-
tative results of this assay to the inhibition of mammalian 5-LOX, it
has been shown that inhibition of plant LOX activity by NSAIDs is
qualitatively similar to their inhibition of the rat mast cell LOX and
may be used as a simple qualitative screen for such activity. (Table
1) Perusal of LOX % inhibition values shows that compound 7
[X ¼ (CH2)2] is the most active, within the set, followed by
compounds 5 and 6. Compounds 16 and 19 (adducts of LA and
compounds 8 and 12) present lower LOX inhibition value compared
to compounds 8 and 12. On the contrary compound 17 (LA’s hybrid
of compound 9) presents 33% LOX inhibition (compound 9 does not
present any inhibition under the reported experimental condi-
tions). The majority of the LOX inhibitors referred in literature acts
as: a) antioxidants or free radical scavengers [48], since lip-
oxygenation occurs via a carbon-centered radical, b) inhibitors to
reduce Fe3þ at the active site to the catalytically inactive Fe2þ (LOXs
contain a ‘‘non-heme’’ iron per molecule in the enzyme active site
as high-spin Fe2þ in the native state and the high-spin Fe3þ in the
activated state) and c) excellent ligands for Fe3þ. In case of
compounds 5–7 their LOX inhibition is correlated with their high
hydroxyl radical scavenging activity.
tests. In view of the differences among the test systems available,
the results of a single assay can give only a suggestion on the
protective potential of tested compounds. Among the plethora of
methods used for the evaluation of the antioxidant activity, the
DPPH test is very useful in the micromolar range demanding
minutes to hours for both lipophilic and hydrophilic samples. In the
cases where the structure of the electron donor is not known this
method can afford data on the reduction potential of the sample,
and hence can be helpful in comparing the reduction potential of
unknown materials. The competition of the compounds with
DMSO for hydroxyl radicals has been used, whereas antioxidant
capability as well as lipid peroxidation inhibitory activity was
assayed by the inhibition of the oxidizing enzyme lipoxygenase
[45].
The interaction of the examined compounds with the stable free
radical DPPH was studied (Table 2). This interaction indicates their
radical scavenging ability in an iron-free system using a stable free
radical which can accept an electron or hydrogen atom [45]. In
general, compounds 5, 6, 8, 9, 16 and 17 were found to have very
low activity, whereas compound 10 showed the highest interaction
(90%) at 0.1 mM, followed by compounds 13, 19, 12 and 11. Not
many changes in interaction % values were observed between the
amides and the corresponding LA hybrids. The adduct 17 and
compound
9 (a coumarin-3-carboxamide derivative) present
4. Conclusions
almost equal interaction values. Compound 12 presents lower
activity than the corresponding adduct 19. Introduction of the
lipoic acid moiety improves the DPPH % interaction with compound
19. For most of the compounds the interaction was not time and
concentration dependent. The time course of DPPH interaction, as
affected by two different concentrations is given in Table 2. In
general, this interaction expresses the reducing activity of the
tested compounds and indicates their ability to scavenge free
radicals.
In the present study, as a first step, we have successfully
developed a synthetic route for the preparation of novel hybrids of
coumarins and lipoic acid. Their synthesis is almost simple with
satisfactory yields. We have then shown that the synthesized
compounds, both the aminoamides and the final hybrids, possess
significant anti-inflammatory activity in vivo and also that 16 and
19 are potent ꢀOH scavengers. Moreover, an overview of the results
of the in vivo experiment reveals that the tested coumarin–lipoic
acid hybrids 16 and 19 exhibit significantly higher activity than
lipoic acid. Also adducts 16 and 19, seem to be more potent in vivo
than the corresponding aminoamides 8 and 12.
The in vivo anti-inflammatory activity of 12 and 19 seems to be
related with their high HOꢀ scavenging and reducing activities,
whereas the high competition of compound 16 with DMSO for
hydroxyl radicals is correlated with its in vivo potency. More
specifically, it is evident that, coumarin aminoamides 11–13 exhibit
satisfactory combined antioxidant–anti-inflammatory activity
therefore the design of this type of dual acting molecules should be
further explored based on the structural features of these
compounds. Among the final hybrids, the hybrid adducts 16 and 19
Lipophilicity does not seem to play a significant role under the
reported experimental conditions.
During the inflammatory process, phagocytes generate the
superoxide anion radical at the inflamed site. Superoxide anion and
H2O2 are the Reactive Oxygen Species (ROS) primarily formed by
phagocytic cells. At sites of inflammation superoxide anion
participates in reaction producing H2O2, the highly reactive
hydroxyl radical and possibly singlet 1O2. When superoxide dis-
mutase comes in contact with the superoxide, it reacts with it and
forms H2O2 which is dangerous in the cell because it can easily
transform into a hydroxyl radical (via reaction with Fe2þ Fenton
chemistry) one of the most destructive free radicals [46]. Hydroxy

3024
G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
present the highest in vivo activity from all the tested compounds,
they are more effective than the corresponding aminoamides 8 and
12 therefore worth further study.
J7,8 ¼ 7.9 Hz, 1H, H-7), 8.15–8.18 (dd, J5,7 ¼ 1.5 Hz, J5,6 ¼ 8.1 Hz, 1H,
H-5), 11.4 (br s, 1H, CONH); Anal. Calcd for C16H12N2O4: C, 64.86, H,
4.08, N, 9.45. Found C, 64.68, H, 4.21, N, 9.63.
5. Experimental section
5.1.5. N-(2-aminoethyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-
3-carboxamide (11)
Melting points were determined on a Galenkamp MFB-595
melting point apparatus and are uncorrected. NMR spectra were
White solid, mp 252–256 ꢂC (dec). Obtained from the reaction of
3-ethoxycarbonyl-4-hydroxy-6-methyl coumarin (6) with 1,2-eth-
recorded on
a
Varian Gemini 2000, 300 MHz spectrometer.
ylenediamine. Yield: 65%; 1H NMR (CDCl3/CF3COOD):
d
¼ 2.48 (s,
Elemental analyses were recorded on a Euro EA3000 Series Euro
Vector CHNS Elemental Analyser.
3H, CH3), 3.46 (br s, 2H, CH2NH2), 3.83 (t, J ¼ 6.9 Hz, 2H, NHCH2),
7.29 (d, J7,8 ¼ 8.2 Hz, 1H, H-8), 7.62 (dd, J5,7 ¼ 1.2 Hz, J7,8 ¼ 8.2 Hz 1H,
H-7), 7.88 (d, J5,7 ¼ 1.2 Hz, 1H, H-5), 9.78 (br s, 1H, CONH); Anal.
Calcd for C13H14N2O4: C, 59.54, H, 5.38, N, 10.68. Found C, 59.77, H,
5.41, N, 10.35.
5.1. General procedure for the synthesis of coumarin-3-
carboxamides
3-Ethoxycarbonyl 4-hydroxycoumarins 5, 6 (1 mmol) were
mixed with the appropriate diamine (2 mmol) in dry toluene (5 mL).
The mixture was stirred at 110 ꢂC for 4 h. The precipitate formed was
filtered off, washed with diethylether and dried in vacuo.
5.1.6. N-(8-aminooctyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-
3-carboxamide (12)
White solid, mp 221–224 ꢂC (dec). Obtained from the reaction 3-
ethoxycarbonyl-4-hydroxy-6-methyl coumarin (6) with 1,8-octa-
methylenediamine. Yield: 98%; 1H NMR (CDCl3/CF3COOD):
5.1.1. N-(2-aminoethyl)-4-hydroxy-2-oxo-2H-chromene-3-
carboxamide (7) [49]
d
¼ 1.34–1.39 (m, 8H, (CH2)2(CH2)4(CH2)2), 1.6–1.69 (m, 4H,
CH2(CH2)4CH2), 2.47 (s, 3H, CH3), 3.15 (t, J ¼ 6.7 Hz, 2H, CH2NH2),
3.45 (t, J ¼ 6.9 Hz, 2H, NHCH2), 7.3 (d, J7,8 ¼ 8.0 Hz, 1H, H-8), 7.59 (d,
J7,8 ¼ 7.9 Hz, 1H, H-7), 7.87 (s, 1H, H-5), 9.81 (br s, 1H, CONH); Anal.
Calcd for C19H26N2O4: C, 65.88, H, 7.57, N, 8.09. Found C, 65.79, H,
7.64, N, 8.21.
White solid, mp 245–247 ꢂC. Obtained from the reaction of
3-ethoxycarbonyl-4-hydroxycoumarin (5) with 1,2-ethylenedi-
amine. Yield: 79%; 1H NMR (CDCl3/CF3COOD)
d
¼ 3.47 (br s, 2H,
CH2NH2), 3.79–3.86 (t, J ¼ 4.8 Hz, 2H, NHCH2), 7.41 (d,
J7,8 ¼ 8.4 Hz, 1H, H-8), 7.48 (pseudotriplet, J ¼ 7.6 Hz, 1H, H-6,),
7.81 (pseudotriplet, J ¼ 8.1 Hz, 1H, H-7), 8.09 (dd, J5,6 ¼ 7.8 Hz,
J5,7 ¼ 1.4 Hz, 1H, H-5), 9.64 (br s, 1H, CONH); Anal. Calcd for
C12H12N2O4: C, 58.06, H, 4.87, N, 11.28. Found C, 58.32, H, 4.73,
N, 11.31.
5.1.7. N-(2-aminophenyl)-4-hydroxy-6-methyl-2-oxo-2H-
chromene-3-carboxamide (13)
Green solid, mp 196–199 ꢂC (dec). Obtained from the reaction
of 3-ethoxycarbonyl-4-hydroxy-6-methyl coumarin (6) with 1,2-
phenylenediamine. Yield: 48%; 1H NMR (CDCl3/CF3COOD):
d
¼ 2.46
5.1.2. N-(6-aminohexyl)-4-hydroxy-2-oxo-2H-chromene-3-
carboxamide (8) [50]
White solid, mp 212–214 ꢂC. Obtained from the reaction of
3-ethoxycarbonyl-4-hydroxycoumarin (5) with 1,6-hexamethy-
(s, 3H, CH3), 6.8 (m, 2H, CONHCCCHCHCCNH2), 7.12 (m, 1H,
CONHCCCCCHCNH2), 7.29 (m, 1H, CONHCCHCCCCNH2), 7.43–7.53
(m, 2H, H-7, H-8), 7.85 (s, 1H, H-5), 10.91 (s, 1H, CONH); Anal. Calcd
for C17H14N2O4: C, 65.80, H, 4.55, N, 9.03. Found C, 66.04, H, 4.38,
N, 9.18.
lenediamine. Yield: 65%; 1H NMR (CDCl3/CF3COOD)
d
¼ 1.15–1.49
(m, 8H, CH2(CH2)4CH2), 2.75 (br s, 2H, CH2NH2), 3.31 (br s, 2H,
NHCH2), 7.06–7.45 (m, 2H, H-6, H-8), 7.62 (pseudotriplet,
J ¼ 7.9 Hz, 1H, H-7), 7.78 (d, J ¼ 8.0 Hz, 1H, H-5), 10.28 (br, 1H,
CONH); Anal. Calcd for C16H20N2O4: C, 63.14, H, 6.62, N, 9.20.
Found C, 63.26, H, 6.67, N, 9.34.
5.2. Synthesis of the final compounds: general procedure
Coumarin-3-aminocarboxamides 7–13 (0.46 mmol) were
mixed with N-(lipoyloxy)succinimide (14) (0.14 g, 0.46 mmol) in
dichloromethane (3 mL) and drops of DMF were added to facili-
tate dissolution of the reactants. The mixture was stirred and
light-protected overnight. Drops of H2O were added to the
mixture and afterwards it was extracted with dichloromethane
(3 ꢃ10 mL) and washed with H2O. The organic extracts were
dried (Na2SO4) and concentrated in vacuo to afford the final
compounds which were triturated with diethylether and filtered
off.
5.1.3. N-(8-octylamino)-4-hydroxy-2-oxo-2H-chromene-3-
carboxamide (9)
White solid, mp 219–222 ꢂC. Obtained from the reaction of 3-
ethoxycarbonyl-4-hydroxycoumarin (5) with 1,8-octamethylene-
diamine. Yield: 71%; 1H NMR (CDCl3/CF3COOD):
d
¼ 1.24–1.45 (m,
8H, (CH2)2(CH2)4(CH2)2), 1.67–1.72 (m, 4H, CH2(CH2)4CH2), 3.14 (t,
J ¼ 6.7 Hz, 2H, CH2NH2), 3.46 (t, J ¼ 6.9 Hz, 2H, NHCH2), 7.37 (dd,
J7,8 ¼ 8.1 Hz, J6,8 ¼ 1.2 Hz, 1H, H-8), 7.46 (dtd, J6,8 ¼ 1.2 Hz,
J6,7 ¼ 7.7 Hz, J5,6 ¼ 7.8 Hz, 1H, H-6), 7.77 (dtd, J5,7 ¼ 1.4 Hz,
J6,7 ¼ 7.7 Hz, J7,8 ¼ 8.1 Hz, 1H, H-7), 8.09 (dd, J5,7 ¼ 1.4 Hz,
J5,6 ¼ 7.8 Hz, 1H, H-5), 9.42 (br s, 1H, CONH); Anal. Calcd for
C18H24N2O4: C, 65.04, H, 7.28, N, 8.43. Found C, 65.27, H, 7.15, N,
8.21.
5.2.1. N-(2-(4-(1,2-dithiolan-3-yl)butanamido)ethyl)-4-hydroxy-2-
oxo-2H-chromene-3-carboxamide (15)
Yellowish solid, mp 151–153 ꢂC. Obtained from the reaction of
N-(2-aminoethyl)-4-hydroxy-2-oxo-2H-chromene-3-carboxamide
(7) with N-(lipoyloxy)succinimide (14). Yield: 75%; 1H NMR
(CDCl3):
d
¼ 1.41–1.5 (m, 2H, CH2CH2CH2CHS), 1.61–1.72 (m, 4H,
5.1.4. N-(2-aminophenyl)-4-hydroxy-2-oxo-2H-chromene-3-
carboxamide (10)
CH2CH2CH2CHS), 1.89 (sextet, J ¼ 6.6 Hz, 1H, SCH2CHH), 2.17–2.23
(m, 2H, NHCOCH2), 2.44 (sextet, J ¼ 5.9 Hz,1H, SCH2CHH), 3.06–3.17
(m, 2H, CH2S), 3.48–3.65 (m, 5H, NH(CH2)2NH, CHS), 6.00 (br, 1H,
NHCO), 7.33–7.4 (m, 2H, H-6, H-8), 7.69 (dtd, J7,8 ¼ 7.5 Hz,
J7,6 ¼ 7.6 Hz, J7,5 ¼1.5 Hz, 1H, H-7), 8.04 (dd, J5,6 ¼ 7.8 Hz,
J5,7 ¼ 1.5 Hz, 1H, H-5), 9.41 (br s, 1H, CONH), 17.67 (s, 1H, OH); Anal.
Calcd for C20H24N2O5S2: C, 55.03, H, 5.54, N, 6.42. Found C, 54.83, H,
5.42, N, 6.56.
Green solid, mp >280 ꢂC. Obtained from the reaction of 3-
ethoxycarbonyl-4-hydroxycoumarin (5) with 1,2-phenylenedi-
amine. Yield: 44%; 1H NMR (CDCl3/CF3COOD):
d
¼ 7.46–7.49 (d,
J7,8 ¼ 7.9 Hz, 1H, H-8), 7.54 (br s, 1H, aromatic), 7.56–7.6 (pseudo-
triplet, J ¼ 7.9 Hz, 1H, H-6), 7.58–7.63 (br s, 1H, aromatic), 7.70–7.78
(m, 2H, aromatics), 7.84–7.89 (dtd, J5,7 ¼ 1.5 Hz, J6,7 ¼ 7.7 Hz,

G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
3025
5.2.2. N-(6-(5-(1,2-dithiolan-3-yl)pentanamido)hexyl)-4-hydroxy-
2-oxo-2H-chromene-3-carboxamide (16)
5.3.1.2. Inhibition of the carrageenin-induced edema [29]. Edema
was induced in the right hind paw of Fisher 344 rats (150–200 g) by
the intradermal injection of 0.1 mL 2% carrageenin in water. Both
sexes were used. Females pregnant animals were excluded. Each
group was composed of 6–15 animals and the experiment was
repeated twice. The animals, which have been bred in our labora-
tory, were housed under standard conditions and received a diet of
commercial food pellets and water ad libitum during the mainte-
nance but they were entirely fasted during the experiment period.
Our studies were in accordance with recognised guidelines on
animal experimentation.
The tested compounds 0.01 mmol/kg body weight, were sus-
pended inwater, with few drops of Tween 80 and ground in a mortar
before use and were given intraperitoneally simultaneously with
the carrageenin injection. The rats were euthanized 3.5 h after
carrageenin injection. The difference between the weights of the
injected and uninjected paws was calculated for each animal. The
change in paw weight was compared with that in control animals
(treated with water) and expressed as a percent inhibition of the
edema CPE % values (Table 1). Indomethacin in 0.01 mmol/kg (47%),
was used as a reference compound. Each value represents the mean
obtained from 6 to 15 animals in two independent experiments with
a standard error of the mean less than 10% (Table 1).
Yellowish solid, mp 128–134 ꢂC. Obtained from the reaction of
N-(6-aminohexyl)-4-hydroxy-2-oxo-2H-chromene-3-carboxamide
(8) with N-(lipoyloxy)succinimide (14). Yield: 50%; 1H NMR (CDCl3)
d
¼ 1.33–1.71 (m, 14H, NHCH2(CH2)4CH2NH, (CH2)3CHS), 1.9 (sextet,
J ¼ 6.5 Hz, 1H, SCH2CHH), 2.14–2.19 (m, 2H, NHCOCH2), 2.45 (sextet,
J ¼ 6.5 Hz, 1H, SCH2CHH), 3.06–3.28 (m, 4H, (CH2)4CH2NH, CH2S),
3.45 (q, J ¼ 6.6 Hz, 2H, NHCH2(CH2)4), 3.56 (q, J ¼ 7.2 Hz, 1H, CHS),
5.47 (br, 1H, NHCO), 7.26–7.39 (m, 2H, H-6, H-8), 7.69 (dd,
J7,5 ¼1.8 Hz, J7,6 ¼ 8.2 Hz, 1H, H-7), 8.05 (dd, 1H, H-5, J5,6 ¼ 8.2 Hz,
J5,7 ¼ 1.8 Hz), 9.26 (br, 1H, CONH), 18.18 (s, 1H, OH); Anal. Calcd for
C24H32N2O5S2: C, 58.51, H, 6.55, N, 5.69. Found C, 58.62, H, 6.59, N,
5.51.
5.2.3. N-(8-(5-(1,2-dithiolan-3-yl)pentanamido)octyl)-4-hydroxy-
2-oxo-2H-chromene-3-carboxamide (17)
Yellow solid, mp 112–117 ꢂC. Obtained from the reaction of N-(8-
aminooctyl)-4-hydroxy-2-oxo-2H-chromene-3-carboxamide (9)
with N-(lipoyloxy)succinimide (14). Yield: 59%; 1H NMR (CDCl3):
d
¼ 1.29–1.73 (m, 18H, NHCH2(CH2)6CH2NH, (CH2)3CHS), 1.9 (sextet,
J ¼ 6.6 Hz, 1H, SCH2CHH), 2.17 (t, J ¼ 7.1 Hz, 2H, NHCOCH2), 2.46
(sextet, J ¼ 6.6 Hz, 1H, SCH2CHH), 3.06–3.19 (m, 2H, CH2S), 3.23 (q,
J ¼ 6.7 Hz, 2H, (CH2)6CH2NH), 3.43 (q, J ¼ 6.3 Hz, 2H, NHCH2(CH2)6),
3.56 (quintet, J ¼ 7.2 Hz, 1H, CHS), 5.47 (br,1H, NHCO), 7.32–7.38 (m,
2H, H-6, H-8), 7.66 (dtd, J7,8 ¼ 7.2 Hz, J7,6 ¼ 7.2 Hz, J7,5 ¼1.6 Hz, 1H,
H-7), 8.04 (dd, J5,6 ¼ 8.0 Hz, J5,7 ¼ 1.6 Hz, 1H, H-5,), 9.26 (br, 1H,
CONH), 18.23 (s, 1H, OH); Anal. Calcd for C26H36N2O5S2: C, 59.97, H,
6.97, N, 5.38. Found C, 60.21, H, 6.77, N, 5.46.
5.3.2. Experiments in vitro
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than
10% of the mean.
5.3.2.1. Determination of the reducing activity of the stable radical
1,1-diphenyl-picrylhydrazyl (DPPH) [29]. To a solution of DPPH in
absolute ethanol (final concentration 0.05 mM), the tested
compounds in final concentration 0.1 and 0.5 mM, dissolved in
ethanol, were added. As control solution ethanol was used. The
concentrations of the solutions of the compounds were 0.1 and
0.5 mM. After 20 and 60 min at room temperature the absorbance
was recorded at 517 nm (Table 2). Nordihydroguaiaretic acid
(NDGA) was used as a reference compound.
5.2.4. N-(2-(4-(1,2-dithiolan-3-yl)butanamido)ethyl)-4-hydroxy-
6-methyl-2-oxo-2H-chromene-3-carboxamide (18)
Yellow solid, mp 106–109 ꢂC. Obtained from the reaction of
N-(2-aminoethyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-3-
carboxamide (11) with N-(lipoyloxy)succinimide (14). Yield:
52%; 1H NMR (CDCl3):
d
¼ 1.4–1.8 (m, 6H, (CH2)3CHS), 1.89
(sextet, J ¼ 6.5 Hz, 1H, SCH2CHH), 2.17–2.23 (m, 2H, NHCOCH2),
2.37–2.49 (m, 4H, CH3, SCH2CHH), 3.06–3.18 (m, 2H, CH2S),
3.47–3.62 (m, 5H, NH(CH2)2NH, CHS), 6.12 (br, 1H, NHCO), 7.2–
7.26 (m, 1H, H-8), 7.48 (d, J ¼ 8.1 Hz, 1H, H-7), 7.79 (s, 1H, H-5),
9.41 (br, 1H, CONH), 17.61 (s, 1H, OH); Anal. Calcd for
C21H26N2O5S2: C, 55.98, H, 5.82, N, 6.22. Found C, 55.71, H,
5.93, N, 6.31.
5.3.2.2. Competition of the tested compounds with DMSO for
hydroxyl radicals [29,52,53]. The hydroxyl radicals were generated
by mixing the Fe3þ/ascorbic acid system. The methyl radical is
produced by trapping the hydroxyl radical with DMSO. One part of
this methyl radical is then changed to formaldehyde. Using this
principal, we determined the production of hydroxyl radical
according to Nash [52,53]. The reaction mixture contained EDTA
5.2.5. N-(8-(5-(1,2-dithiolan-3-yl)pentanamido)octyl)-4-hydroxy-
6-methyl-2-oxo-2H-chromene-3-carboxamide (19)
Yellowish solid, mp 139–141 ꢂC. Obtained from the reaction of
N-(8-aminooctyl)-4-hydroxy-6-methyl-2-oxo-2H-chromene-3-
carboxamide (12) with N-(lipoyloxy)succinimide (14). Yield: 33%;
(0.1 mM), Fe3þ (167
mM), DMSO (33 mM) in phosphate buffer
(50 mM, pH 7.4), the tested compounds (concentration 0.1 mM)
and ascorbic acid (10 mM). After 30 min of incubation (37 ꢂC) the
reaction was stopped with CCl3COOH (17% w/v) (Table 1) and the
formaldehyde formed was detected spectrophotometrically at 412.
1H NMR (CDCl3):
d
¼ 1.3–1.7 (m, 18H, NHCH2(CH2)6CH2NH,
(CH2)3CHS), 1.9 (sextet, J ¼ 6.7 Hz, 1H, SCH2CHH), 2.17 (t, J ¼ 7.5 Hz,
2H, NHCOCH2), 2.39–2.48 (m, 4H, CH3, SCH2CHH), 3.06–3.17 (m, 2H,
CH2S), 3.23 (q, J ¼ 6.6 Hz, 2H, (CH2)6CH2NH), 3.43 (q, J ¼ 6.6 Hz, 2H,
NHCH2(CH2)6), 3.56 (quintet, J ¼ 7.1 Hz,1H, CHS), 5.5 (br,1H, NHCO),
7.2-7.26 (m, 1H, H-8), 7.46 (d, J ¼ 8.1 Hz, 1H, H-7), 7.81 (s, 1H, H-5),
9.27 (br, 1H, CONH), 18.16 (s, 1H, OH); Anal. Calcd for C27H38N2O5S2:
C, 60.65, H, 7.16, N, 5.24. Found C, 60.82, H, 7.28, N, 5.09.
5.3.2.3. Soybean lipoxygenase inhibition study in vitro [29]. In vitro
study was evaluated as reported previously. The tested compounds
dissolved in ethanol were incubated at room temperature with
sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution
(1/9 ꢃ 10ꢁ4 w/v in saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor (Table 1).
5.3. Biological experiments
5.3.1. Experiments in vivo
Acknowledgement
5.3.1.1. Acute toxicity tests. Rats were divided into 5 groups (6–8
animals each), in which different doses (0.1–0.5 mmol/kg body
weight) of the test compounds at the lethal range were injected.
Mortality was recorded 24 h post-injection.
We would like to thank Dr. C. Hansch and Biobyte Corp. 201
West 4th Str., Suite 204, Claremont CA California, 91711, USA for free
access to the C-QSAR program [51].

3026
G. Melagraki et al. / European Journal of Medicinal Chemistry 44 (2009) 3020–3026
[26] U. Galm, A. Dessoy, J. Schmidt, L.A. Wessjohann, L. Heide, Chem. Biol. 11 (2004)
References
173.
[27] J. Tao, S. Hu, M. Pacholec, C.T. Walsh, Org. Lett. 5 (2003) 3233.
[28] S.D. Larsen, J.R. Colca, P.K.W. Harris, H.R. Miller, A.L. Laborde, R.F. Kletzien,
H.J. Schostarez, Patent WO 97/18808, CAN 127:60619, 1997.
[29] A. Detsi, D. Bouloumbasi, K.C. Prousis, M. Koufaki, G. Athanasellis,
G. Melagraki, A. Afantitis, O. Igglessi-Markopoulou, C. Kontogiorgis,
D.J. Hadjipavlou-Litina, J. Med. Chem. 50 (2007) 2450.
[30] G.Ph. Biewenga, G.R.M.M. Haenen, A. Bast, Gen. Pharmacol. 29 (1997) 315.
[31] L. Packer, E.H. Witt, H.J. Tritschler, Free Radical Biol. Med. 19 (1995) 227.
[32] H. Moini, L. Packer, N.-E.L. Saris, Toxicol. Appl. Pharmacol. 182 (2002) 84.
[33] H. Gurer, H. Ozgunes, S. Oztezcan, N. Ercal, Free Radical Biol. Med. 27 (1999)
75.
[34] R. Alleva, E. Nasole, F. Di Donato, B. Borghi, J. Neuzil, M. Tomasetti, Biochem.
Biophys. Res. Commun. 333 (2005) 404.
[35] M. Koufaki, T. Calogeropoulou, A. Detsi, A. Roditis, A.P. Kourounakis,
P. Papazafiri, K. Tsiakitzis, C. Gaitanaki, I. Beis, P.N. Kourounakis, J. Med. Chem.
44 (2001) 4300.
[36] M. Rosini, V. Andrisano, M. Bartolini, M.L. Bolognesi, P. Hrelia, A. Minarini,
A. Tarozzi, C. Melchiorre, J. Med. Chem. 48 (2005) 360.
[37] M.M. Ponpipom, R.L. Bugianesi, T.J. Blake, J. Med. Chem. 30 (1987) 705.
[38] M. Koufaki, A. Detsi, E. Theodorou, C. Kiziridi, T. Calogeropoulou,
A. Vassilopoulos, A.P. Kourounakis, E. Rekka, P.N. Kourounakis, C. Gaitanaki,
P. Papazafiri, Bioorg. Med. Chem. 12 (2004) 4835.
[39] G. Athanasellis, G. Melagraki, H. Chatzidakis, A. Afantitis, A. Detsi, O. Igglessi-
Markopoulou, J. Markopoulos, Synthesis 11 (2004) 1775.
[40] T.Y. Shen, Nonsteroidal antiinflammatory agents, in: M.E. Wolf (Ed.), Burger’s
Medicinal Chemistry, John Wiley and Sons, New York, 1980, pp. 1217–1219.
[41] C.A. Winter, in: S. Gattini, M.N.G. Dukes (Eds.), Nonsteroidal Antiinflammatory
Drugs, Excepta Medica, Amsterdam, 1965, p. 190.
[1] B. Halliwell, R. Aeschbach, J. Loliger, O.I. Aruoma, Food Chem. Toxicol. 33
(1995) 601.
[2] G. Garrido, D.G. Lez, C. Del porte, N. Backhouse, G. Quinterno, A.J. Nunez-Selles,
M.A. Morales, Phytother. Res. 15 (2001) 18.
[3] R. O’Kennedy, R.D. Thornes, Coumarins. Biology, Applications and Mode of
Action, Wiley, New York, 1997.
[4] J.R.S. Hoult, M. Paya, Gen. Pharmacol. 27 (1996) 713.
[5] L.M. Bedoya, M. Beltran, R. Sancho, D.A. Olmedo, S. Sanchez-Palomino, E. del
Olmo, J.L. Lopez-Perez, E. Munoz, A. San Feliciano, J. Alcamı, Bioorg. Med.
Chem. Lett. 15 (2005) 4447.
[6] K.-H. Lee, J. Nat. Prod. 67 (2004) 273.
[7] S. Thaisrivongs, P.K. Tomich, K.D. Watenpaugh, et al., J. Med. Chem. 37 (1994)
3200.
[8] S. Kirkiacharian, D.T. Thuy, S. Sicsic, R. Bakhchinian, R. Kurkjian, T. Tonnaire, Il
Farmaco 57 (2002) 703.
[9] A.G. Kidane, H. Salacinski, A. Tiwari, K.R. Bruckdorfer, A.M. Seifalian, Bio-
macromolecules 5 (2004) 798.
[10] G. Appendino, E. Mercalli, N. Fuzzati, L. Arnoldi, M. Stavri, S. Gibbons,
M. Ballero, A. Maxia, J. Nat. Prod. 67 (2004) 2108.
[11] K.M. Khan, Z.S. Saify, M.Z. Khan, Zia-Ullah, M.I. Choudhary, Atta-Ur-Rahman,
S. Perveen, Z.H. Chohan, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 19 (2004)
373.
[12] C. Ito, M. Itoigawa, Y. Mishina, V.C. Filho, F. Enjo, H. Tokuda, H. Nishino,
H. Furukawa, J. Nat. Prod. 66 (2003) 368.
[13] B.H. Lee, M.F. Clothier, F.E. Dutton, G.A. Conder, S.S. Johnson, Bioorg. Med.
Chem. Lett. 8 (1998) 3317.
[14] C.A. Kontogiorgis, D. Hadjipavlou-Litina, J. Med. Chem. 48 (2005) 6400.
[15] M. Ghate, D. Manohar, V. Kulkarni, R. Shobha, S.Y. Kattimani, Eur. J. Med. Chem.
38 (2003) 297.
[16] A.M. Silvan, M.J. Abad, P. Bermejo, M. Sollhuber, A. Villar, J. Nat. Prod. 59 (1996)
1183.
[17] D.N. Nicolaides, K.C. Fylaktakidou, K.E. Litinas, D. Hadjipavlou-Litina, Eur. J.
Med. Chem. 33 (1998) 715.
[18] H.G. Raj, V.S. Parmar, S.C. Jain, S. Goel, Pooman, Himanshu, S. Malhotra,
A. Singh, C.E. Olsen, J. Wengel, Bioorg. Med. Chem. 6 (1998) 833.
[19] C.A. Kontogiorgis, D. Hadjipavlou-Litina, Bioorg. Med. Chem. Lett. 14 (2004)
611.
[42] T. Kuroda, F. Suzuki, T. Tamura, K. Ohmori, H. Hosoe, J. Med. Chem. 35 (1992)
1130.
[43] L. Flohe, R. Beckman, H. Giertz, G. Loschen, Oxygen-centered free radicals as
mediators of inflammation, in: H. Sies (Ed.), Oxidative Stress, Academic Press,
London, 1985, pp. 403–435.
[44] L.A. Saldan, G. Elias, M.N.A. Gao, Arzneim-Forsch. Drug Res. 40 (1990) 89.
[45] R. Matsukawa, Z. Dubinsky, E. Kishimoto, K. Masaki, T. Takeuchi, M. Chihara,
Y. Yamamoto, E. Niki, I. Karube, J. Appl. Phys. 9 (1997) 29.
[46] O.I. Aruoma, M. Wasil, B. Halliwell, B. Hoey, J. Butler, J. Biochem. Pharmacol. 36
(1987) 3739.
[20] M. Ghate, R.A. Kusanur, M.V. Kulkarni, Eur. J. Med. Chem. 40 (2005) 882.
[21] I.A. Khan, M.V. Kulkarni, M. Gopal, M.S. Shahabuddin, C.M. Sun, Bioorg. Med.
Chem. Lett. 15 (2005) 3584.
[22] C.A. Kontogiorgis, D. Hadjipavlou-Litina, J. Enzyme Inhib. Med. Chem. 18
(2003) 63.
[23] A. Maltese, F. Maugeri, K.W. Ward, C. Bucolo, Biomed. Chromatogr. 21 (2007)
351.
[24] Y.K. Tyagi, A. Kumar, H.G. Raj, P. Vohra, G. Gupta, R. Kumari, P. Kumar,
R.K. Gupta, Eur. J. Med. Chem. 40 (2005) 413.
[47] R.B. Taraborewala, J.M. Kauffman, J. Pharm. Sci. 79 (1990) 173.
[48] K. Muller, Arch. Pharmacol. 3 (1994) 327.
[49] J.F. Harred, A.R. Knight, J.S. McIntyre, Inventors, Dow Chemical Company,
Assignee. Epoxidation process. U.S. Patent 3654317, 1972 Apr 4.
[50] J. Deng, N. Neamati, J.M. Briggs, J. Med. Chem. 49 (2006) 1684.
[51] Biobyte Corp., C-QSAR Database 201. West 4th Str., Suite 204, Claremont CA,
California 91711, USA.
[52] T. Nash, Biochem. J. 55 (1953) 416.
[53] S.M. Klein, G. Cohen, A.I. Cederbaum, Biochemistry 20 (1981) 6006.
[25] Z.-Q. Liu, W. Yu, Z.-Li. Liu, Chem. Phys. Lipids 103 (1999) 125.