Synthesis and antiinflammatory activity of coumarin derivatives

Article abstract of DOI:10.1021/jm0580149

The synthesis of several coumarin Mannich bases is described. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity was determined experimentally by RPTLC method. All compounds were evaluated fo

Full text of DOI:10.1021/jm0580149

6400
J. Med. Chem. 2005, 48, 6400-6408
Synthesis and Antiinflammatory Activity of Coumarin Derivatives†,‡
Christos A. Kontogiorgis and Dimitra J. Hadjipavlou-Litina*
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki,
54124, Thessaloniki, Greece
Received March 1, 2005
The synthesis of several coumarin Mannich bases is described. The structures of the synthesized
compounds were confirmed by spectral and elemental analysis. Their lipophilicity was
determined experimentally by RPTLC method. All compounds were evaluated for their
antiinflammatory and antioxidant activity and for their ability to inhibit in vitro lipoxygenase.
The derivatives were found to present antioxidant and antiinflammatory activities. The tested
derivatives inhibited carraggeenin-induced hind paw edema. They also significantly suppressed
the arthritis induced by Freund’s adjuvant. Compound 10, the most active in vivo, was found
to possess protective properties against adjuvant-induced arthritis in rats. The biological in
vitro activities were concentration dependent. Hydrophilicity, the presence of a free 7-OH, and
steric requirements for the substituent at position 8 are the most important factors in terms
of SAR. An attempt was made to correlate several physicochemical properties of the molecules
with their in vivo/in vitro activity.
Introduction
Inflammation is a complex phenomenon involving
interrelationships humoral and cellular reactions through
a number of inflammatory mediators. It is a usual
symptom covering different pathologies, and there are
still many questions to be answered in order to under-
stand the inflammatory process as well as a need for
better-tolerated and more efficient nonsteroidal antiin-
flammatory drugs. In the pathways of the inflammatory
process, the implication of free radicals is particularly
important. It has also been reported that antiinflam-
matory drugs may be effective in the prevention of free
radical-mediated damage.1
Coumarins have been reported to have multiple
biological activities.2 They have been used to treat such
diverse ailments as cancer, burns, brucellosis, and
cardiovascular and rheumatic diseases.3 The coumarin
molecule has been shown to possess unique antiedema
and antiinflammatory activities. Thus, coumarin de-
rivatives could be particularly effective in the treatment
of all high protein edemas.4-6 Various coumarin-related
derivatives are recognized as inhibitors of the lipoxy-
Figure 1.
genase and cyclooxygenase pathways of arachidonate
metabolism7-9 but also of neutrophile-dependent super-
oxide anion generation.10 Several natural or synthetic
coumarins with various hydroxyl and other substituents
were found to inhibit lipid peroxidation and to scavenge
hydroxyl radicals and superoxide anion11 and to influ-
ence processes involving free radical-mediated injury,
as can some plant phenolics and flavonoids.12
It has been presented earlier that coumarin Mannich
bases play a significant role as biologically active
compounds in various diseases (Figure 1, structures
a-c].13-15 Mannich bases were also found to act as
antiinflammatory agents (Figure 1, structure d).16-18
Thus, we found it interesting to synthesize some
coumarin Mannich bases as possible antiinflammatory
agents to examine (a) their ability to inhibit various
enzymes involved in the arachidonic acid cascade and
(b) their antioxidant behavior. The structure of our
derivatives combines the biological active group of 7-OH-
coumarin and the amine moiety as a new template for
antiinflammatory activity. The suggested structural
variations could affect both efficacy and their tolerability
partly due to differences in their physicochemical prop-
* To whom correspondence should be addressed. Phone: +30231-
0997627, Fax: +302310997679, e-mail: hadjipav@pharm.auth.gr.
† This article is dedicated to Professor D. Nicolaides, Laboratory of
Organic Chemistry, Department of Chemistry, Aristotle University of
Thessaloniki, Greece.
‡ A part of this work has been presented in the 3rd European
Graduate Student Meeting, Feb 23-25, 2001, Frankfurt, Germany;
International Summer School for Postdoctoral Scientists and Advanced
Students by NATO/FEBS Advanced Study Instute-INTAS Symposium,
Aug 18-30, 2002, Spetses, Greece; 3rd Panhellenic Congress of Free
Radicals and Oxidative Stress, Oct 3-5, 2002, Athens, Greece; 20th
Greek Medical Military Congress, Nov 18-21, 2004, Thessaloniki,
Greece.
10.1021/jm0580149 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/13/2005

Antiinflammatory Coumarin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6401
Table 1. Chemical Data of Coumarin Derivatives
Scheme 1. Synthesis of Compounds 2-13
erties, which determine their distribution in the body
and their ability to pass through and to enter the
interior membranes.19,20
Today there is an increased interest in the develop-
ment of effective nonacidic antiinflammatory agents,
since the currently used acidic NSAIDs cause develop-
ment of untoward side effects in a significant fraction
of people.21,22
Chemistry. The synthesis of the coumarin deriva-
tives 2-13 (Table 1) was accomplished according to the
Mannich reaction as indicated in Scheme 1. Examples
of 7-hydroxycoumarin condensed with formaldehyde and
amines have been reported previously, for example,
compounds 3 and 11,23,24 which were found to possess
potent stimulating activity on the nervous and respira-
tory system. The synthesis of compound 8 was given in
a previous publication.15 We have included them in our
study in order to delineate structure-activity relation-
ships, within these Mannich bases.
In our case 7-hydroxycoumarin, formaldehyde, and
the corresponding amine, e.g. primary or secondary, or
alicyclic, benzyl-, or phenethylamines in 1:1:1 molar
ratio, respectively,13 were refluxed and stirred in abso-
lute ethanol for 8-12 h. The reactions were monitored
by thin-layer chromatography. In the case of compound
2 (R ) CH3) an oxazine ring is formed including 7-O
(from 7-OH) and 8-C (from the phenyl ring). Compounds
3-13 have been identified as 7-hydroxy-8-substituted-
aminomethyl coumarin derivatives.
In all cases it was assumed that, as in formylation,
the Claisen rearrangement and the Fries shift13 with
7-hydroxycoumarin, the new substituent would appear
at position 8. This indicates that the 8- substituent next
to the 7-hydroxy plays a detrimental role in determining
the course of the reaction.
In some cases the purification of the new compounds
was very difficult. Some derivatives were crystallized

6402 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Kontogiorgis and Hadjipavlou-Litina
Table 2. Lipophilicity Values: Experimentally Determined RM
and Theoretically Calculated Clog P Values, Inhibition % of
Induced Carrageenin Rat Paw Edema (CPE %), and in Vitro
at the right hind paw in comparison to the uninjected
left hind paw. Carrageenin- induced edema is a non-
specific inflammation resulting from a complex of
diverse mediators.28 Since edemas of this type are highly
sensitivetononsteroidalantiinflammatorydrugs(NSAIDs),
carrageenin has been accepted as a useful agent for
studying new antiinflammatory drugs.29 This model
reliably predicts the antiinflammatory efficacy of the
NSAIDs, and during the second phase it detects com-
pounds that are antiinflammatory agents as a result of
inhibition of prostaglandin amplification.30 As shown in
Table 2, all the investigated compounds induced protec-
tion against carrageenin-induced paw edema. The pro-
tection ranged from 31.2 to 77.7% while the reference
drug, indomethacin, induced 47% protection at an
equivalent concentration. Compounds 10 and 11 were
the most potent (75.7 and 77.7%) whereas compound 4
had the lowest effect (31.2%). A slight increase in
inhibition values was observed (compounds 5, 6, 7, and
8, Table 2) with the dose increase from 0.01 to 0.1 mmol.
The nature and the presence of the alicyclic amines
(piperazinyl, morpholinyl ring) in compounds 10 and 11
(Table 2) seem to be correlated with the higher inhibi-
tion values. The ionization constants of these com-
pounds might play a significant role. Both compounds
possess low lipophilicity, 1.11 and 1.32 (Table 2). It
seems that hydrophilicity (low lipophilicity values)
partly affect the antiinflammatory activity, as it can be
concluded from the following equation.
Inhibition of Soybean Lipoxygenase (LOX) (IC50
)
CPE (%)c,d
0.01 mmol/kg LOXe IC50 µM
compd Clog Pa
RMb ((SD)
body weight
(% 0.1 mM)
1
1.62 -0.46 (( 0.02) nof
43 ( 6 (99.6)
2
1.37 -0.20 (( 0.02) 53.6** ( 2. 4 42 ( 0.6 (100)
3
4
2.60 -0.50 (( 0.01) 47.4* ( 1.4
3.04 -0.43 (( 0.02) 31.2* (1.9
3.48 -0.38 (( 0.02) 52.7* ( 1.8
2.82 -0.39 (( 0.02) 55.4* ( 2.6
5.64 -0.16 (( 0.01) 46.2** ( 2.2 13 ( 0.5 (99.0)
3.22 -0.42 (( 0.017) 47.5** ( 3.1 44 ( 1 (100)
1.47 -0.33 (( 0.02) 53.6** ( 1.7 52 ( 0.7 (100)
1.11 -0.44 (( 0.01) 77.7* ( 1.4
1.32 -0.44 (( 0.01) 75.7* ( 1.5
37 ( 0.2 (98.8)
41 ( 0.03 (99.6)
59 ( 4 (83.3)
47 ( 2 (85.7)
5g
6h
7i
8j
9
10
11
12
13
100 ( 5 (51.6)
.1000 (26.4)
.1000 (40.7)
.1000 (13.8)
0.76
0.04 (( 0.001) 56.0* ( 2.1
0.42 NDk
53.4* ( 2.9
coumarin nt
nt
nt
nt
30.2** ( 1.8 - (15.1)
warfarin
nt
nt
41.0* ( 2.5
no
e
NDGAe
nt
a Theoretically calculated clog P values. b RM values are the
average of at least 10 measurements. c Each value represents the
mean of two independent experiments with five animals in each
group; statistical studies were done with student’s T-test, * p <
0.01, ** p < 0.05. d Indomethacin as a standard 47% (0.01 mM).
e Nordihydroguaiaretic acid (NDGA) 83.7% (0.1 mM), 94.7% (1
mM). f no: no action under the experimental conditions. g % CPE
at 0.1 mmol/kg 54.7% ((1.3). h % CPE at 0.1 mmol/kg 58.6%
((2.9). i % CPE at 0.1 mmol/kg 47.5%. j % CPE at 0.1 mmol/kg
52.7%; (ED50 0.055 mmoles/kg bw). k ND: not determined unter
the reported experimental conditions; nt: not tested.
as salts (hydrobromic acid was used for 5, 6, 7, 8, and
9 and hydrochloric acid for 2), recrystallized by absolute
ethanol, and decomposed upon melting. Most of the
derived bases are very soluble in absolute ethanol (2,
3, 5, 6, 7, 8, 9). The structures of the compounds are
confirmed by UV, IR, 1H NMR, 13C NMR, MS and
elemental analysis.
Physicochemical Studies. Since lipophilicity is a
significant physicochemical property determining dis-
tribution, bioavailability, metabolic activity, and elimi-
nation, we tried to determine experimentally from
RPTLC25 the lipophilicity of the synthesized derivatives
as RM values and to compare them with the correspond-
ing to theoretically calculated Clog P values26,27 in
n-octanol-buffer (Table 1).
From our results (Table 1) it can be concluded that
RM values could not be used as a successful relative
measure of the overall lipophilic/hydrophilic balance of
these molecules. We could attribute this to the different
nature of the hydrophilic and lipophilic phases in the
two systems and to the presence of basic nitrogen atoms
in the examined compounds, which could disturb the
absorption/desorption process.
% CPE ) - 0.101 (( 0.079) Clog P +
1.881 (( 0.142)
n ) 10, r ) 0.723, r2 ) 0.523, q2 ) 0.170,
s ) 0.085, F1,7 ) 8.6, a ) 0.01
Higher lipophilicity does not seem to affect the inhibi-
tory activity, since compound 7 with Clog P 5.64 inhibits
the carrageenin-induced edema at a range of 46.2%.
Compounds 10 and 11 have been chosen to be
examined on adjuvant-induced disease (AID) since both
highly inhibited the carrageenin paw edema. Adjuvant-
induced disease (AID) is a good experimental model of
rheumatoid arthritis and is often used for testing agents
for antiinflammatory activity. Rats treated with com-
pounds 10 and 11 did not develop severe arthritis,
indicating that these coumarin derivatives exhibited
possible immunomodulating activity, an activity to be
further confirmed. The time course of adjuvant arthritis
development, expressed as arthritic scores, is shown in
Table 3. Arthritic score, body weight loss, cahexia, and
in vivo metabolism impairment (expressed as the dura-
tion of the induced paralysis) were significantly reduced
in the experiment (Table 4) (Figure 2). Compounds were
found to influence the body weight. The percent change
of the body weight for compound 10 was 18.4% and for
compound 11 was 7%. In the same experimental pro-
tocol phenylbutazone (80 mg/ kg po) gave an arthritic
score of 1.5 (for controls, the arthritic score was 9.0) on
the 24th day after the administration of FA (Table 3).25
The duration of zoxazolamine-induced paralysis was
reduced to normal levels. Although, N-alkyl-7-ami-
nocoumarins are substrates of cytochrome P450-isoen-
zymes,31 the present data require further investigation
regarding the mechanism of action, to delineate that the
Discussion
In this investigation, we synthesized some novel
coumarin Mannich bases that were expected to offer
protection against inflammation and radical attack, by
application of standard synthetic methods summarized
in Scheme 1.
In acute toxicity experiments, the in vivo examined
compounds did not present toxic effects in doses up to
0.5 mmol/kg body weight.
The in vivo antiinflammatory effects of the tested
coumarins were assessed by using the functional model
of carrageenin-induced rat paw edema and are pre-
sented in Table 2, as the percentage of weight increase

Antiinflammatory Coumarin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6403
Table 3. Effect of Compounds 1, 10, 11 on the Onset (day of
appearance after Freund’s Adjuvant Injection, FA) and Severity
of Arthritis (arthritic scores) in Adjuvant-Induced Disease
(AID) Rats
activity are both a free 7-OH group on the phenyl ring
and an alicyclic amine (piperazinyl or morpholinyl ring)
or a primary aliphatic amine. This is in accordance with
previous findings, where Mannich bases were found to
be potent antiinflammatory agents.15,16
days after
FA’s
AID rats
AID rats
AID rats
treated with
treated with treated with
AID rat
injection compound 1a compound 10a compound 11a controlsa
Compounds were further evaluated for inhibition of
soybean lipoxygenase LOX by the UV absorbance-based
enzyme assay.32 While one may not extrapolate the
quantitative 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 2). Perusal of IC50 values shows that compound
7 is the most active, within the set, followed by
compounds 3 and 4. Most of the LOX inhibitors are
antioxidants or free radical scavengers,33 since lipoxy-
genation occurs via a carbon-centered radical. Some
studies suggest a relationship between LOX inhibition
and the ability of the inhibitors to reduce Fe3+ at the
active site to the catalytically inactive Fe2+. LOXs
contain a “non-heme” iron per molecule in the enzyme
active site as high-spin Fe2+ in the native state and the
high spin Fe3+ in the activated state. Several LOX
inhibitors are excellent ligands for Fe3+. Many fla-
vonoids and other phenolics such as hydroxycoumarin
derivatives inhibit soybean lipoxygenase. 7-Hydroxy-
coumarin also inhibit the activity of LOX.6 This inhibi-
tion is related to their ability to reduce the iron species
in the active site to the catalytically inactive ferrous
form.32,33 Thus the presence of a free 7-OH group could
explain the inhibition results of the examined com-
pounds. Although lipophilicity is referred36 as an im-
portant physicochemical property for LOX inhibitors, all
the above tested derivatives do not follow this concept
with one exception. Compound 7 shows the highest log
P value 5,64 and it is highly potent.
0
2
0
0
0
0
0
0
0
0
4
0
0
0
0
6
8
0
0
0
0
0
0
0
0
10
12
14
16
18
20
22
24
0
0
0
0
0
0
0
0
0
0
0
6.6
8.1
11.8
14.0
10.8
8.8
1.2
1.8
2.6
5.2
4
0
1.25
2.5
3.25
4.25
3.5
3.2
3
4.8
5.8
a Number of arthritic scores (SD less than 10%); phenylbutazone
80 mg/kg po gave arthritic score of 0.3 (for controls the arthritic
score was 9.0) on the 24th day.
Figure 2. The number of arthritic scores of rats treated with
compounds 1, 10, 11 compared to rat controls.
hepatoprotective effect of compounds 10 and 11 during
inflammation could be mediated via the ability of
compound to induce drug-metabolizing enzymes.
Compound 1 (7-hydroxycoumarin) was also tested in
the same experiment in order to delineate the role of
the 7-hydroxycoumarin in this biological response. From
our results it seems that rats treated with 7-hydroxy-
coumarin did not develop arthritis; the arthritic score,
body weight loss, cahexia, and the in vitro metabolic
impairment is significantly reduced (Tables 3 and 4,
Figure 2). Unfortunately the rats treated with com-
pound 1 presented side effects from the gastrointestinal
route (ulcers and hyperemia). On the contrary these
effects were not observed after the treatment with
compounds 10 and 11, since both are not acidic.
COX-1 plays a role as a “housekeeping enzyme”, for
example maintaining the lining of the stomach’s endo-
thelial cells and contributing to the normal function of
the cardiovascular system via the release of prostacy-
clin. Thus, inhibition of COX-1 is involved in the
appearance of the unwanted side effects. Compounds
10 and 11, which possess the highest in vivo activity
against the CPE test and no side effects from the
gastrointestinal route, were also examined for their
inhibitory activity against COX-1 at 1 mM (Table 6).
Both compounds did not highly inhibit COX-1 (11 and
32%).
In this investigation all compounds were studied in
order to gain insight in the mechanism of their an-
tiphlogistic action. It is well-known that free radicals
play an important role in inflammatory process.37
Concerning the structural features of the active
coumarin Mannich bases, our data indicate that the
minimal structural requirements for antiinflammatory
Table 4. Assessment of the Preventive Action of 10 on the Adjuvant-Induced Disease (AID) Manifestations (body weight change,
inflammation-arthritic score, zoxazolamine paralysis)
absolute controls,
normal animals,
treated with the
AID rat controls
treated only with
examined parameters
AID rats
AID rats
AID rats
(mean ( SD)
treated with 1 treated with 10 treated with 11 the liquid vehicle liquid vehicle only
percent change of body weight (g ( SD)
18.6 ( 8.4
18.4 ( 5.9
7.0 ( 4.2
-14.8 ( 4.5
-18 ( 4.4
inflammation (arthritic scores)b
4 (8.8)
5.8 (8.8)
3.5 (8.8)
8.8
no arthritic scores
are observed
218 ( 15.5
zoxazolamine paralysis (min ( SD)
232.5 ( 1.4
292 ( 18.8
212.5 ( 1.7
347.0 ( 4.9
a Counted on the 24th day after Freund’s Adjuvant (FA) injection; figures in parentheses denote score for controls.

6404 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Kontogiorgis and Hadjipavlou-Litina
Table 5. Interaction % with DPPH (RA %).a Competition % with DMSO for Hydroxyl Radical (HO• %)b
RA (%) 20 min
0.2 mM
RA (%) 60 min
0.2 mM
HO• (%)
0.01 mM
compd
0.1 mM
0.5 mM
0.1 mM
0.5 mM
0.1 mM
1
2
3
4
5
6
7
8
9
1.5
9.1
2.8
11.3
6.3
8.3
19.8
22.7
58.5
26.0
24.0
20.0
21.0
21.5
26.4
81.3
48.6
6.2
2.4
14.8
10.7
20.7
19.0
21.0
22.0
26.4
12.5
17.5
41.8
31.7
11.2
82.6
nt
5.6
16.2
12.0
33.4
22.8
26.0
21.0
39.4
14.6
15
8.9
28.3
33.9
67.8
24.8
27.0
25.0
37.0
20.8
30.6
89.0
55.1
8.7
nt
11.3
1.9
nt
37.2
46.9
39.0
81.1
78.4
77.8
51.5
88.0
75.9
81.1
69.8
90.9
nt
5.3
12.6
18.0
20.0
18.0
23.0
13.7
16.4
35.0
26.6
13.9
81
24.1
22.0
25.0
17.0
29.0
18.0
17.0
51.6
28.3
9.2
80
nt
52.7
nt
nt
nod
13.3
27.5
22.1
30.2
65.7
60.9
75.9
27.5
38
10
11
58.4
30.2
11.0
80
12
13
NDGAa
trolox
BHTb
coumarin
warfarin
96.5
nt
75.4
5.8
10.3
96.5
nt
98.2
6.7
12.8
nt
73.4
nt
nt
nt
ntc
nt
88.2
nt
78.0
95.8
31.3
4.9
60
78
21
24
nt
9.2
nt
a NDGA (nordihydroguaiaretic acid) b BHT (butylated hydroxytoluene). c nt: not tested. d no: no action under the experimental
conditions.
Table 6. In Vitro Inhibition of Cyclooxygenase-1 (COX-1%)
(58.5%) and 12 (48.6%). For most of the compounds the
interaction was time and concentration dependent. The
time course of DPPH interaction, as affected by various
concentrations, is given in Table 5. In general, this
interaction expresses the reducing activity of the tested
compounds and indicates their ability to scavenge free
radicals. It was found that the ethylenediamine deriva-
tive, as well as the morpholinyl and the phenethylamine
were the most potent in interacting with DPPH.
compd
COX-1%, 1 mM
10
11 ( 0.3
32 ( 1.3
57.8 ( 4.3
11
Sc-560
Table 7. % In Vitro Lipid Peroxidation (LP %). % Superoxide
Radical Scavenging Activity (PMS %)
LP (%)
Preliminary QSAR studies on the values of DPPH
interaction have shown that the sterimol properties,39
e.g. L, the length of the first atom of the substituent
R1, R2 groups plays a significant role. It seems that the
smaller L leads to higher % interaction values.
compd
0.1 mM
1 mM
PMS (%), 1 mM
1
2
3
4
5
6
7
8
9
14.6
noa
no
18.2
8.6
no
no
19.5
no
19.5
19.5
no
no
9.1
no
26.1
no
80.6
86.1
no
22.2
no
7.6
5.5
5.7
During the inflammatory process, phagocytes gener-
ate the superoxide anion radical at the inflamed site,
and this is connected to other oxidizing species such as
HO•. Hydroxyl radicals are produced by reactions that
depend on transition metals, particularly iron.40 Hy-
droxy radicals are among the most reactive oxygen
species and are considered to be responsible for some
of the tissue damage occurring in inflammation. Hy-
droxy 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 syn-
thesis.
The competition of coumarins with DMSO for HO•
generated by the Fe3+/ascorbic acid system, expressed
as percent inhibition of formaldehyde production, was
used for the evaluation of their hydroxyl radical scav-
enging activity. In these experiments (Table 5) com-
pounds 3 and 4 did not show any inhibition at 0.01 mM.
Compounds 2 and 5 (0.01 mM) slightly inhibited the
oxidation of DMSO (33 mM). Compounds 6, 7, 8 showed
mild inhibition at 0.01 mM. The inhibition was found
to increase as the concentration of the tested compounds
was increased. Compound 13 (90% at 0.1 mM) was the
most active under these experimental conditions, whereas
the “mother compound” coumarin presents 78% com-
petition at the high concentration (0.1 mM), showing
that the coumarin nucleus by itself had scavenging
activity. The order of decreasing HO• scavenging activity
no
33.3
no
13.7
35.6
5.5
no
no
10
21.9
19.9
22.1
ni
19.2
no
no
11
58.3
no
12
13
50.0
88.9
25
86.1
no
coumarin
warfarin
caffeic acid
NDGA
no
no
5.5
26.1
a no: no action show under the experimental conditions
Consequently, compounds with antioxidant properties
could be expected to offer protection in rheumatoid
arthritis and inflammation and to lead to potentially
effective drugs. In fact, many nonsteroidal antiinflam-
matory drugs have been reported to act either as
inhibitors of free radical production or as radical scav-
engers.38 Thus, we tested these derivatives with regard
to their antioxidant ability and in comparison to well-
known antioxidant agents, e.g. nordihydriguaiaretic
acid, trolox, caffeic acid (Tables 5 and 7).
The interaction of the examined compounds with the
stable free radical DPPH was studied (Table 5). This
interaction indicates their radical scavenging ability in
an iron-free system. Compounds 2, 3, 9, and 13 were
found to have very low activity, followed by compounds
5, 6, 7, 8, 10, and 12. Compound 11 showed the highest
interaction (81%) at 0.5 mM, followed by compounds 4

Antiinflammatory Coumarin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6405
was 13 > 9 > 5-11 > 6 > 7 > 10 > 12 > 8 > 3. The
overall low lipophilicity is correlated with higher com-
petition values. Antioxidants of hydrophilic or lipophilic
character are both needed to act as radical scavengers
in the aqueous phase or as chain-breaking antioxidants
in biological membranes. The most potent compound 13
possesses the lowest Clog P value within the set.
It seems that LOX % inhibition in vitro is correlated
with the ‚HO• radical scavenging activity as well as with
the DPPH interaction values (% LOX is log % inhibitory
activity on LOX induced by the tested compounds at 1
mM, RA % the log % interaction with DPPH, and % OH
the log % of the competition with DMSO for hydroxyl
radicals at the same concentration).
Experimental Section
Materials. All the chemicals used were of analytical grade
and commercially available from Merck. 1,1-Diphenyl-2-pic-
rylhydrazyl (DPPH) and nordihydroguairetic acid (NDGA)
were purchased from the Aldrich Chemical Co., Milwaukee,
WI. Soybean lipoxygenase, linoleic acid sodium salt arachi-
donic acid (AA), nicotinamidoadenine dinucleotide (NADH),
nitrotetrazolium Blue (NBT), porcine heme, butylated hy-
droxytoluene (BHT), and indomethacin were obtained from
Sigma Chemical Co. (St. Louis, MO) and carrageenin, type K,
was commercially available. For the in vivo experiments, male
and female Fischer-344 rats (180-240 g) were used, and the
kit for COX activity assay was purchased from Cayman.
N-Methylphenazonium-mehtyl sulfate was purchased by Flu-
ka. Freund’s Adjuvant (FA) referred to 0.6 mg desiccated
Mycobacterium butyricum was suspended in 0.1 mL liquid
paraffin.
% LOX ) 2.070 (( 1.061) % RA +
Synthesis All starting materials were obtained from com-
mercial sources and used without further purification. Melting
Points (uncorrected) were determined on a MEL-Temp II (Lab.
Devices, Holliston, MA). UV-vis spectra were obtained on a
Perkin-Elmer 554 double beam spectrophotometer and on a
Hitachi U-2001 spectrophotometer. Infrared spectra (film as
Nujol mulls) were recorded with Perkin-Elmer 597 spectro-
photometer (The Perkin-Elmer Corporation Ltd., Lane Bea-
consfield, Bucks, England) and a Shimadzu FTIR-8101M. The
1H nucleic magnetic resonance (NMR) spectra were recorded
at 300 MHz on a Bruker AM 300 spectrometer (Bruker
Analytische Messtechnik GmbH, Rheinstetten, Germany) in
CDCl3 or DMSO using tetramethylsilane as an internal
standard unless otherwise stated. 13C NMR spectra were
obtained at 75.5 MHz on a Bruker AM 300 spectrometer in
CDCl3 or DMSO solutions with tetramethylsilane as internal
reference unless otherwise stated. Mass spectra were deter-
mined on a VG-250 spectrometer (VG-Labs., Tritech, England)
with ionization energy maintained at 70 eV. Elemental
analyses were obtained on an acceptable range ((0.4%) in a
Perkin-Elmer 240B CHN analyzer (The Perkin-Elmer Corpo-
ration Ltd.). Reactions were monitored by thin-layer chroma-
tography (TLC) by Fluka, on aluminum cards precoated with
0.2 mm of silica gel and fluorescent indicator.
3.033 (( 1.954)% OH - 6.753 (( 4.906) (2)
n ) 9, r ) 0.890, r2 ) 0.792, q2 ) 0.596, s )
0.168, F2,9 ) 11.36, R ) 0.01
The tested compounds did not highly inhibit the lipid
peroxidation. In some cases a small inhibition increase
is observed by increasing their concentration, e.g.
compound 1, 8, 10, 12 (Table 7). Nonenzymatic super-
oxide anion radicals were generated. Compounds 1, 2,
11, and 13 present high scavenging activity (Table 7).
7-Hydroxycoumarin, the “mother molecule”, seems to
strongly scavenge superoxide radicals. In general, lipo-
philicity does not positively influence the scavenging
activity.
We tried to linearly correlate the expressions of
antiinflammatory, antioxidant, free radical scavenging
activity, and LOX inhibition activity for all the tested
compounds. None of these correlations were satisfactory
enough (r < 0.6). Presumably, these activities proceed
via at least partially different mechanisms.
General Procedure. The reaction was performed according
to the literature9-11,30,31 after modification. To a solution of the
appropriate amines (0.005 mol) in 30 mL of absolute ethanol
was added aqueous formaldehyde (37%) (0.01 mol). After 0.5
h of gentle refluxing at 60 °C, the 7-hydroxycoumarin (1) was
added (0.005 mol), dissolved in 30 mL of absolute ethanol. The
duration of the reaction was 8-12 h, and the reaction was
monitored by TLC. The solvent was removed under reduced
pressure.
Attempts to correlate these expressions of activity
with RM values in a linear or nonlinear regression
analysis gave statistically nonsignificant correlations.
Unfortunately the number of compounds is not enough
to calculate a combination of all the effects.
Conclusion
The present study has shown that certain coumarin
Mannich bases possess high antiinflammatory activity.
Their synthesis is almost simple with satisfactory yields.
Most of them are potent HO• scavengers and inhibit in
vitro soybean lipoxygenase. Hydrophilicity, the presence
of a free 7-OH, and steric requirements for the sub-
stituent at position 8 are the most important factors in
terms of SAR. Although the antiinflammatory mecha-
nism explaining the activity on CPE remains unclear,
the in vivo antiinflammatory activity of the synthesized
compounds seems to be related with their high HO•
scavenging and reducing activity, in vitro.
Two compounds, 10 and 11, were found to present a
promising antiinflammatory profile: combining immu-
nomodulating high antiedematous ability with no side
effects from the gastrointestinal route and significant
inhibitory activity on LOX. These compounds constitute
an interesting template for the evaluation of new
synthetic lipoxygenase inhibitors and may be helpful for
the design of new therapeutic tools against inflamma-
tion.
Gerneral Procedure for Salts Preparation. A. A cold
alcohol solution of the product was treated with concentrated
hydrochloric acid, and the hydrochloride was obtained. The
salt was purified by absolute ethanol/ethyl ether.
B. Following the procedure of Burke et al.,41 to a solution
of the reaction mixture in propanol-1 (85% propanol-1) was
added hydrobromic acid in ethanol absolute. After 30 min of
gentle refluxing at 60 °C and cooling, ethyl ether was added
dropwise. Upon cooling and filtration, the product was ob-
tained.
3-Methyl-3,4,4a,10a-tetrahydro-2H-1,5-dioxa-3-aza-
phenanthren-6-one (2). According to the general procedure,
7-hydroxycoumarin was reacted with methylamine in absolute
ethanol. Following the general procedure for salt preparation
A, the hydrochloride was obtained. (12%, salt). The salt
decomposes upon melting. UV (ethanol absolute) λmax: 348,
279, 236, ꢀmax: 1795, 1935, 2132; IR (Nujol) (cm-1): 1710-
1720 (CdO), 1250 (C-N); 1H NMR (DMSO-d6, CDCl3): δ 7.72
(d, J ) 10.2, 1H), 7.47 (d, J ) 8.9, 1H), 7.18 (d, J ) 8.9, 1H),
6.22 (d, J ) 10.2, 1H), 4.65 (br, s, 3H), 4.55 (br, s, 2H), 2.83
(br, 3H); 13C NMR (DMSO-d6, CDCl3): 165.7, 153.7, 145.8,
143.9, 126.6, 113.6, 112.6, 112.1, 107.8, 84.5, 46.9, 40.0; MS:
218 [M+], 217 (35.7), 174 (41.5), 146 (100), 90 (43.8), 89 (39.2);
Anal.: (C12H12ClNO3) C, H, N.

6406 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Kontogiorgis and Hadjipavlou-Litina
7-Hydroxy-8-piperidin-1-ylmethyl-chromen-2-one (3).
According to the general procedure, 7-hydroxycoumarin was
reacted with piperidine in absolute ethanol. The product was
collected and recrystallized from ethanol (52%): mp 173-175
°C; UV (ethanol absolute) λmax: 382, 307, 237, ꢀmax: 2175, 1886,
2082; IR (Nujol) (cm-1): 3450-3300 (O-H), 1720-1700 (Cd
O), 1300(C-N); 1H NMR (CDCl3): δ 9.41 (s, 1H), 7.61 (d, J )
8.9, 1H), 7.26 (d, J ) 8.9, 1H), 6.73 (d, J ) 8.9, 1H), 6.17 (d, J
hydrobromide was made according to the general procedure
for salt preparation B. The product was recrystallized with
methanol and ether (16%, salt): mp 222-225 °C; UV (ethanol
absolute) λmax: 348, 302, 238, ꢀmax: 1713, 1837, 2053; IR (Nujol)
(cm-1): 3400-3300 (O-H), 1720-1730 (CdO), 1280 (C-N);
1H NMR (DMSO-d6, CDCl3): δ 9.28 (s, 1H, disappears in
addition of D2O), 7.96 (d, J ) 8.9, 1H), 7.67-7.31 (br, 7H),
6.80 (d, J ) 8.9, 1H), 6.28 (d, J ) 8.9, 1H), 4.26 (br-s, 2H),
4.23 (br-s, 2H), 3.35 (br-s, 2H, disappears in addition of D2O);
13C NMR (DMSO-d6, CDCl3): 160.2, 159.6, 154, 144.6, 131.6,
130.3, 128.9, 128.5, 112.4, 111.4, 111.2, 105.4, 50.3, 37.9; MS:
281 M+], 190 (67.7), 175 (60.2), 146 (70.0), 106 (90.8), 91 (100);
Anal: (C17H16NO3Br) C, H, N.
8-Ethylaminomethyl-7-hydroxy-chromen-2-one (9). Ac-
cording to the general procedure, 7-hydroxycoumarin was
reacted with ethylamine in absolute ethanol. Following the
general procedure for salt preparation B, the hydrobromide
was obtained. (22% salt); mp 236-237 °C; UV (ethanol
absolute) λmax: 338, 259, ꢀmax: 3771, 3766; IR (Nujol) (cm-1):
3400-3300 (O-H), 1730-1720 (CdO), 1250 (C-N); 1H NMR
(DMSO-d6, CDCl3): δ 11.36 (br-s, 1H), 7.72 (d, J ) 10.17, 1H),
7.47 (d, J ) 5.1, 1H), 7.19 (d, J ) 8.91, 1H), 6.23 (d, J ) 5.1,
1H), 4.22 (br-s, 2H), 3.36 (br-s, 2H), 3.04 (br-s, 2H), 1.26 (m,
3H); 13C NMR (DMSO-d6, CDCl3): 160.0, 159.7, 154.0, 144.7,
130.5, 113.1, 112.3, 111.3, 105.4, 53.7, 37.8, 22.4; MS 219 [M+]
(35), 176 (100), 146 (74), 118 (55), 91 (53); Anal.: (C12H14NO3-
Br) C, H, N.
) 8.9, 1H), 4.0 (s, 2H), 2.9-2.2 (br, 4H), 1.8-1.5 (br, 6H); 13
C
NMR (CDCl3): 163.4, 161.4, 153.1, 144.3, 143.9, 127.8, 113.9,
111, 107.7, 54.3, 53.8, 25.6, 23.6; MS: 259 [M+], 175 (36), 146
(24), 98 (26), 84 (100); Anal.: (C15H17NO3) C, H, N.
7-Hydroxy-8-(phenethylamino-methyl)-chromen-2-
one (4). According to the general procedure, 7-hydroxycou-
marin was reacted with phenethylamine in absolute ethanol.
The product was collected and recrystallized from ethanol. The
product was collected and recrystallized from ethanol (36%):
mp 120-121 °C; UV (ethanol absolute) λmax: 390, 305, 239,
ꢀmax: 2308, 1890, 2089; IR (Nujol) (cm-1): 3400-3300 (O-H,
1
N-H), 1720-1710 (CdO), 1250 (C-N); H NMR (CDCl3): δ
7.61-7.58 (d, J ) 8.91, 1H), 7.33-7.22 (m, 7H), 6.78-6.75 (d,
J ) 8.91,1H), 6.18-6.15 (d, J ) 8.91, 1H), 4.30 (br, 2H), 4.17-
3.44 (br, 1H), 3.03 (tr, J ) 6.4, 2H), 2.91 (tr, J ) 6.4, 2H); 13
C
NMR (CDCl3): 161.5, 161.4, 153.6, 144.2, 143.9, 129.3, 128.7,
128.4, 126.9, 114.1, 111.9, 111.5, 101.1, 49.0, 34.8, 33.5; MS:
295 [M+], 204 (43), 175 (70), 145 (18), 91 (100); Anal. (C18H17-
NO3) C, H, N.
3-Pentyl-3,4-dihydro-2H-1,5-dioxa-3-aza-phenanthren-
6-one (5). The reaction followed the general procedure using
1-pentylamine. The hydrobromide was made according to the
general procedure for salt preparation B (8% salt): mp 198-
201 °C; UV (ethanol absolute) λmax: 347, 278, 240, ꢀmax: 1730,
1862, 2056; IR (Nujol) (cm-1): 3450-3300 (O-H), 1730-1720
7-Hydroxy-8-piperazine-1-ylmethyl-chromen-2-one (10).
According to the general procedure, 7-hydroxycoumarin was
reacted with piperazine in absolute ethanol. The product was
collected and was recrystallized with dichloromethane and
ether (35%): mp 231-233 °C; UV (ethanol absolute) λmax: 322,
219, ꢀmax: 2079, 2034; IR (Nujol) (cm-1): 3450 (O-H), 3050
(N-H), 1730-1720 (CdO); 1H NMR (DMSO-d6, CDCl3): δ 9.42
(s, 1H), 7.62 (d, J ) 9.5, 2H), 7.30 (d, J ) 8.3, 1H), 6.78 (d, J
) 8.3, 1H), 6.20 (d, J ) 9.5, 1H), 4.09 (s, 2H), 2.72-2.96 (br,
9H); 13C NMR (DMSO-d6, CDCl3): 163.6, 162.4, 154.5, 145.6,
129.7, 115.2, 112.9, 109.0, 108.7, 54.7, 53.8, 52.3; MS: 260
[M+], 175 (64.9), 105 (37.7), 85 (63); Anal.: (C14H16N2O3) C,
H, N.
1
(CdO),1250 (C-N); H NMR (DMSO-d6, CDCl3): δ 11.20 (s,
1H), 8.78 (m, 2H), 7.89 (d, J ) 9.4, 1H), 7.55 (d, J ) 8.5, 1H),
7.03 (d, J ) 8.5, 1H), 6.23 (d, J ) 9.4, 1H), 4.28 (s, 2H), 3.08-
2.92 (m, 2H), 1.86-1.69 (m, 2H), 1.60-1.05 (m, 4H), 0.92-
0.79 (br, s, 3H); 13C NMR (DMSO-d6, CDCl3): 160.2, 159.7,
154.1, 144.3, 130.3, 112.6, 111.7, 111.3, 105.0, 47.3, 38.5, 28.3,
25.0, 21.8, 13.7; MS: 261 [M+], 204 (38.6), 175 (100), 57 (95.7);
Anal.: (C15H20BrNO3) C, H, N.
3-Isobutyl-3,4-dihydro-2H-1,5-dioxa-3-aza-phenanthren-
6-one (6). According to the general procedure, 7-hydroxycou-
marin was reacted with isobutylamine absolute ethanol. The
hydrobromide was made according to the general procedure
for salt preparation B (12% salt): mp 232-234 °C; UV (ethanol
absolute) λmax: 321, 209, ꢀmax: 1743, 3757; IR (Nujol) (cm-1):
3450-3300 (O-H), 1720-1710 (CdO), 1250 (C-N); 1H NMR
(DMSO-d6, CDCl3): δ 11.6 (s, 1H), 8.01 (d, J ) 9.09, 1H), 7.65
(d, J ) 10.92, 1H), 7.43 (d, J ) 9.12, 1H), 6.3 (d, J ) 9.09,
1H), 4.46 (s, 2H), 3.37-3.25 (s, 2H), 3.02-3.00 (br-s, 2H), 2.36-
2.22 (br, 2H), 0.98 (s, 6H); 13C NMR (DMSO-d6, CDCl3): 160.2,
159.2, 154.3, 144.5, 131.0, 112.3, 111.6, 111.3, 103.6, 48.7, 45.5,
36.1, 23.8; MS: 247 [M+] (59.3), 204 (79.4), 175 (96.3), 146
(57.9), 100.24 (96.8); Anal. (C14H18BrNO3) C, H, N.
7-Hydroxy-8-morpholin-4-ylmethyl-chromen-2-one (11).
According to the general procedure, 7-hydroxycoumarin was
reacted with morpholine in absolute ethanol. The product was
collected and was recrystallized with ethanol absolute and
dried (13%): mp 231-233 °C; UV (ethanol absolute) λmax: 348,
316, 231, ꢀmax: 2040, 2199, 2411; IR (Nujol) (cm-1): 3450-
3300 (O-H), 3200-3150 (N-H), 1720-1710 (CdO), 1250 (C-
1
N); H NMR (DMSO-d6, CDCl3): δ 9.42 (s, 1H), 7.86 (d, J )
9.84, 1H), 7.47 (d, J ) 8.85, 1H), 6.93 (d, J ) 8.85, 1H), 6.17
(d, J ) 9.84, 1H), 3.7-4.1 (br, 6H), 2.50-2.60 (m, 4H); 13C NMR
(DMSO-d6, CDCl3): 160.2, 159.4, 153.6, 144.5, 127.6, 112.7,
112.4, 111.4, 106.6, 61.9, 53.1, 50.3; MS 261 [M+], 175 (9.4),
87 (93); Anal.: (C14H15NO4) C, H, N.
8-[(2-Aminoethylamino)-methyl]-7-hydroxy-chromen-
2-one (12). According to the general procedure, 7-hydroxy-
coumarin was reacted with 1,2-diamine-ethane in absolute
ethanol. The product was collected and was recrystallized with
ethanol absolute and dried (43%): mp: 215-217 °C; UV
(ethanol absolute) λmax: 211, 294, ꢀmax: 2934, 721; IR (Nujol)
(cm-1): 3350-3250 (O-H), 3150-3050 (N-H), 1700-1680
8-Dipentylaminomethyl-7-hydroxy-chromen-2-one (7).
The reaction was made according to the general procedure.
Dipentylamine was used. The hydrobromide was made ac-
cording to the general procedure for salt preparation B. The
product was recrystallized from acetone (7%, salt): mp 127-
128 °C; UV (ethanol absolute) λmax: 348, 274, 240, ꢀmax: 1612,
1757,1963; IR (Nujol) (cm-1): 3400-3250 (O-H), 1720-1730
1
(CdO), 1250 (C-N); H- NMR (DMSO-d6, CDCl3): δ 8.02-
8.00 (d, J ) 9.09, 2H), 7.66-7.62 (d, J ) 10.92, 1H), 7.44-
7.43 (d, J ) 9.12, 1H), 6.31-6.28 (d, J ) 9.09, 1H), 3.92-2.88
(br, 4H), 4.30 (s, 2H), 2.18-1.98 (br, 2H), 1.56-1.32 (br, 2H),;
13C NMR (DMSO-d6, CDCl3): 160.1, 159.2, 153.9, 144.7, 133.2,
112.4, 111.5, 111.3, 106.3, 54.6, 44.0, 36.4; MS: 218 (9.6), 144
(14), 130 (20), 55 (100); Anal.: (C12H14N2O3) C, H, N; (C12H15
BrN2O3) C, H, N.
1
(CdO), 1270 (C-N); H NMR (DMSO-d6, CDCl3): δ 11.42 (s,
1H), 7.99 (d, J ) 8.7, 1H), 7.65 (d, J ) 8.7, 1H), 7.01 (d, J )
8.7, 1H), 6.27 (d, J ) 8.7, 1H), 4.39 (s, 2H), 3.35 (s, 2H), 2.92-
3.21 (br, 4H), 2.03-1.74 (m, 4H), 1.53-1.23 (m, 8H), 1.08-
0.79 (m, 6H); 13C NMR (DMSO-d6, CDCl3): 158.8, 157.8, 152.9,
143.0, 129.4, 110.9, 110.1, 109.8, 102.7, 51.5, 42.8, 26.7, 21.1,
20.1, 12.1; MS: 332 [M+], 274 (93.5), 260 (23.4), 175 (86.4),
158 (32.7), 147 (53.6), 100 (95.8); Anal. (C20H30BrNO3) C, H,
N.
8-[(3-Aminopropylamino)-methyl]-7-hydroxy-chromen-
2-one (13). According to the general procedure, 7-hydroxy-
coumarin was reacted with 1,3-diaminopropane in absolute
ethanol. The product was collected and was recrystallized with
ethanol absolute and dried (17%): mp 217-219 °C; UV
(ethanol absolute) λmax: 326, 207, ꢀmax: 652, 887; IR (cm-1):
3-Benzyl-3,4-dihydro-2H-1,5-dioxa-3-aza-phenanthren-
6-one (8). According to the general procedure, 7-hydroxycou-
marin was reacted with benzylamine absolute ethanol. The

Antiinflammatory Coumarin Derivatives
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20 6407
3400-3300 (O-H), 3200-3150 (N-H), 1720-1700 (CdO),
1250 (C-N),; MS: 175 (25.1), 174 (46), 133 (28), 103 (42), 84
(100); Anal.: (C13H16N2O3) C, H, N.
tion of DPPH (0.1 mM) in absolute ethanol was added an equal
volume of the compounds dissolved in ethanol. As control
solution ethanol was used. The concentrations of the solutions
of the compounds were 0.01, 0.02, and 0.05 mM. After 20 and
60 min at room temperature, the absorbance was recorded at
517 nm (Table 5).
Competition of the Tested Compounds with DMSO for
Hydroxyl Radicals.25 The hydroxyl radicals generated by the
Fe3+/ascorbic acid system were detected according to Nash by
the determination of formaldehyde produced from the oxida-
tion of DMSO. The reaction mixture contained EDTA (0.1
mM), Fe3+ (167 `ıM), DMSO (33 mM) in phosphate buffer (50
mM, pH 7.4), the tested compounds (concentration 0.01 mM
and 0.1 mM), and ascorbic acid (10 mM). After 30 min of
incubation (37 °C), the reaction was stopped with CCl3COOH
(17% w/v) (Table 3).
Physicochemical Studies. (a) Determination of lipophi-
licity as RM values. Reversed phase TLC (RPTLC) was
performed on silica gel plates impregnated with 55% (v/v)
liquid paraffin in light petroleum ether.25 The mobile phase
was a methanol/water mixture (75/25, v/v) containing 4%
aqueous ammonia (27%). The plates were developed in closed
chromatography tanks saturated with the mobile phase at 24
°C. Spots were detected under UV light or by iodine vapors.
RM values were determined from the corresponding Rf values
(from 10 individual measurements) using the equation RM
log [(1/Rf) - 1] (Table 2).
)
(b) Determination of lipophilicity as Clog P. Lipophilicity
was theoretically calculated as Clog P values in n-octanol-
buffer by CLOGP Program of Biobyte Corp.27
Soybean Lipoxygenase Inhibition Study in Vitro.25
The 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 (1/3 × 104 w/v in saline). The conversion of
sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was
recorded and compared with the appropriate standard inhibi-
tor (nordihydroguaiaretic acid 0.1 mM 83.7% inhibition; 1 mM
94.7% inhibition) (Table 5).
Biological Experiments. Experiments in Vivo. Toxic-
ity of the Examined Compounds. Toxicity experiments
were carried out using both male and female Fischer 344 rats.
Pregnant females were excluded. The tested compounds were
dissolved in water (salts) or suspended with few drops of
Tween 80 and ground in water and administered by intrap-
eritoneal injection at various concentrations. Mortality was
recorded after 24 h.
In Vitro Cyclooxygenase-1 (COX-1) Inhibition Study.42
Cyclooxygenase (COX) activity was determined by using
arachidonic acid (AA) as substrate and N,N,N,N-tetrameth-
ylphenylenediamine (TMPD) as cosubstrate, as described by
Kulmacz and Lancs.41 The reaction mixture (1 mL) contained
0.75 µM heme, 128 `ıM TMPD, 80 µM AA, and 1.5 µg of enzyme,
in 0.1 M Tris/HCl (pH 8.5). The oxidation of substrate, starter
of the reaction, was measured at 37 °C by monitoring the
increase of absorbance at 611 nm. The absorption due to the
spontaneous oxidation of TMPD was subtracted from the
initial rate of oxidation observed in the presence of AA. The
inhibition of the studied compounds was determined after
preincubation for 6 min with the enzyme in the presence of
heme and TMPD, and the reaction was started by adding AA.
For coumarins, 10 µL of scalar dilutions of the inhibitors in
ethanol/water 1/10 (v/V) was added. Sc-560 has been used as
a comparison COX-1 inhibitor (Table 6).
Inhibition of the Carrageenin-Induced Edema.25 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. Pregnant females were
excluded. Each group was composed of 6-15 animals. The
animals, which have been bred in our laboratory, were housed
under standard conditions and received a diet of commercial
food pellets and water ad libitum during the maintenance, but
they were entirely fasted during the experiment period. Our
studies were in accordance with recognized guidelines on
animal experimentation.
The tested compounds, 0.01 mmol/kg body weight, were
suspended in water with a few drops of Tween 80 and ground
in a mortar before use (bases) or dissolved in water (salts) and
were given intraperitoneally simultaneously. The rats were
euthanized 3.5 h after carrageenin injection. The difference
between the weight 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 2. Indomethacin at 0.01 mmol/kg gave 47%
inhibition. Values CPE % are the mean from two different
experiments with a standard error of the mean less than 10%.
Induction of Adjuvant-Induced Disease (AID).25 Groups
of five animals were used. Rats were divided into three
groups: groups 1 and 2 were injected19 with Freund’s Adjuvant
(FA) intradermally, into the base of the tails of the animals,
and were treated with compound 10, which was found to be
the most active in the carrageenin paw edema test. Compound
10 was injected ip in a dose of 0.001 mmol/kg from day zero
once every other day for the following 24 days. Group 3, used
as an absolute control, was injected with liquid vehicle only.
Adjuvant arthritis was developed ca. 14 days post FA admin-
istration. Arthritic score was measured every 2 days from the
commencement (14th day). For quantification of arthritis
(arthritic score), a single point was assigned for each inflam-
maed wrist or ankle area and an additional point was given
for each involved phalangeal joint, up to a maximum of five
points per extremity (Table 7). On the 24th day and at least
12 h after the last injection, animals were administered
zoxazolamine ip 10 mg/1 mL/g, as an aqueous suspension with
a few drops of Tween 80, and the duration of the paralysis
and the inflammation were assessed on the 24th day (Table
4). The experiment was conducted in duplicated.
Heme Protein-Dependent Lipid Degradation.43 Heme
(50 µM), arachidonic acid (0.4 mM), the compounds at the
various concentrations tested, and H2O2 (0.5 mM) were
incubated together for 10 min at 37 °C in KH2PO4-KOH buffer
(50 mM, pH 7.4). The product of peroxidation was detected
using the TBA test.44 The compounds were added in DMSO
solution, which has no effect on the assay (Table 7).
Nonenzymatic Assay of Superoxide Radicals. Mea-
surement of Superoxide Radical Scavenging Activity.45
The superoxide producing system was set up by mixing
phenazine methosulfate (PMS), NADH, and air (oxygen). The
production of superoxide was estimated by the nitroblue
tetrazolium method. The reaction mixture containing 3 µM
PMS, 78 µM NADH, and 25 µM NBT in 19 µM phosphate
buffer pH 7.4 was incubated for 2 min at room temperature
and the absorption measured at 560 nm against a blank
containing PMS. The tested compounds were preincubated for
2 min before adding NADH (Table 7).
Experiments in Vitro. In the in vitro assays each experi-
ment was performed at least in triplicate, and the standard
deviation of absorbance was less than 10% pf the mean.
Determination of the Reducing Activity of the Stable
Radical 1,1-Diphenyl-picrylhydrazyl (DPPH).25 To a solu-
Acknowledgment. We would like to thank Dr. C.
Hansch and Biobyte Corp., 201 West 4th St., Suite 204,
Claremont, CA 91711, for free access to the C-QSAR

6408 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 20
Kontogiorgis and Hadjipavlou-Litina
Synthesis, anti-inflammatory activity and gastrointestinal toxic-
ity. J. Pharm. Sci. 1992, 149-154.
program. The authors are also grateful to Dr K. Litinas,
Asssociate Professor in Laboratory of Organic Chemis-
try, Aristotle University of Thessaloniki, for his invalu-
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