Synthesis and biological evaluation of some 5-arylidene-2-(1,3-thiazol-2- ylimino)-1,3-thiazolidin-4-ones as dual anti-inflammatory/antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2012.10.046

As a part of our ongoing studies in developing new derivatives as dual antimicrobial/anti-inflammatory agents we describe the synthesis of novel 5-arylidene-2-(1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-ones. All newly synthesized compounds were tested for

Full text of DOI:10.1016/j.bmc.2012.10.046

Bioorganic & Medicinal Chemistry 21 (2013) 532–539
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Bioorganic & Medicinal Chemistry
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Synthesis and biological evaluation of some 5-arylidene-2-(1,3-thiazol-
2-ylimino)-1,3-thiazolidin-4-ones as dual anti-inflammatory/antimicrobial
agents
I. Apostolidis a, K. Liaras a, A. Geronikaki a, , D. Hadjipavlou-Litina , A. Gavalas , M. Sokovic ,
⇑
a
a
b
´
b
b
´
ˇ
´
J. Glamoclija , A. Ciric
a Department of Pharmaceutical Chemistry of Aristotle University of Thessaloniki, School of Pharmacy, Thessaloniki 54124, Greece
b University of Belgrade, Institute for Biological Research ‘S. Stankovi´c’, Mycological Laboratory, Belgrade 11000, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of our ongoing studies in developing new derivatives as dual antimicrobial/anti-inflammatory
agents we describe the synthesis of novel 5-arylidene-2-(1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-ones.
All newly synthesized compounds were tested for their anti-inflammatory activity using carrageenan
mouse paw edema bioassay. Their COX-1/LOX inhibitory activities were also determined. Moreover, all
compounds were evaluated for their antimicrobial and antifungal activities against a panel of Gram posi-
tive, Gram negative bacteria and moulds. All tested compounds exhibited better antimicrobial activity
than commercial drugs, bifonazole, ketoconazole, ampicillin and streptomycin.
Received 14 September 2012
Accepted 31 October 2012
Available online 17 November 2012
Keywords:
Thiazoles
Thiazolidinones
Antimicrobial
Antifungal
Ó 2012 Elsevier Ltd. All rights reserved.
Antiinflammatory
COX/LOX inhibitors
1. Introduction
sign innovative agents with different mode of action so that no
cross-resistance with the present therapeuticals can occur.
Inflammation represents a fundamental host response to a wide
range of stimuli such as trauma, tissue injury, microbial activity,
burns, surgery, sepsis, toxic entities, ischemia–reperfusion, post-
ischemic or autoimmune injury.
While the role of microorganisms in inflammation is well recog-
nized, therapeutic strategies with effects on both the microorgan-
ism and associated inflammation have received considerably less
attention.1 Therapeutic options investigated for inflammation con-
trol include neutralization of tumor-necrosis factor (TNF), blockers
of leukotriene receptors, inhibitors of cytokines or leukotriene syn-
thesis and other principal components of the inflammatory re-
sponse for systemic conditions.
Identification of novel compounds which treat both infectious
and inflammatory states more effectively, and which lack side ef-
fects associated with current therapies, remains a major challenge
in biomedical research.3 The use of several drugs to treat inflam-
matory conditions associated with infection is a problem, espe-
cially in case of patients with impaired liver or kidney function
or to avoid drug–drug interaction. In addition, from the phar-
maco-economic cost-effective standpoint, and seeking for a better
patient compliance, a dual anti-inflammatory/antimicrobial agent
with minimum adverse effects and high safety margin is highly
desirable. This promoted us to try to find out agents that have a
dual effect as anti-inflammatory/antimicrobial agents.
During the past 50 years significant efforts in the diagnosis and
treatment of microbial diseases have been accomplished.2 These
efforts led to impressive gains in the treatment of microbial
diseases introducing a range of therapeutic strategies in clinical
practice. In spite of a large number of antibiotics and chemothera-
peutics available for medical use at the same time the emergence
of old and new antibiotics resistance created in the last decades
a substantial medical need for new classes of antibacterial agents.
A potential approach to overcome the resistance problem is to de-
Thiazole derivatives are known to posses anti-inflammatory,
analgesic and antipyretic activities.4–9 Thiabendazole and 2-(p-
chlorophenyl) thiazole-4-acetic acid are widely used as anti-
inflammatory drugs.10 Meloxicam, for example, is a new NSAID
with a thiazolyl group in its structure.11,12 Furthermore, Nirida-
zole13 and some other thiazole derivatives14–20 have been found
to exhibit antimicrobial antifungal/antihelminthic activities.
As a part of our ongoing studies in developing new active anti-
microbials,17,21–23 we report herein the synthesis of
a new
4-thiazolidinone derivatives incorporating two known bioactive
heterocyclic nuclei such as thiazolyl and thiazolidinonyl9,21–25 in
an attempt to present also anti-inflammatory activities. The
⇑
Corresponding author. Tel.: +30 2310 997616; fax: +30 2310 998559.
E-mail address: geronik@pharm.auth.gr (A. Geronikaki).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.046

I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
533
structural variations were selected by introducing, at the 5 position
of thiazolidinone moiety, different arylidene substituents that we
recently exploited as bioactive, on heterocyclic scaffolds, useful
to encompass certain physico-chemical properties as hydrophobic
and steric.21,22,24
Twenty one new 5-arylidene-2-(1,3-thiazol-2-ylimino)-1,3-
thiazolidin-4-ones (Scheme 1, IV-1-21) were synthesized and
evaluated for their in vitro antimicrobial properties against Gram
positive, Gram negative bacteria and fungi, as well as for their
ability to act as anti-inflammatory agents.
using microdilution test. The minimal inhibitory concentrations
that inhibited the growth of the tested microorganisms (MIC)
and minimal bactericidal/fungicidal concentration (MBC/MFC)
were detected.
The results of antimicrobial testing against a panel of selected
Gram positive and Gram negative bacteria are reported in Table 1,
along with those of reference drugs Ampicillin and Streptomycin.2
The structural modifications to previously synthesized thiazolidi-
nones21 involve the nature of the substituent X as well as R.
All the compounds tested showed strong antibacterial activity
against all the species tested. MIC of compounds was ranged from
0.68–40.6 ꢀ 10ꢁ2
l
mol/ml and MBC at 1.44–46.4 ꢀ 10ꢁ2
lmol/ml.
2. Results and discussion
2.1. Chemistry
The antibacterial potential could be presented as follows:
4h > 4j > 4s > 4i > 4k > 4e > 4c > 4f > 4g > 4l > 4m > 4r > 4q > 4t >
4u > 4p > 4o > 4b > 4d. The best antibacterial effect was observed
for compound 4h with inhibitory activity at 0.73–2.56 ꢀ
The compounds described in this paper were synthesized by the
multi-step reaction protocol reported earlier by us.21,22
10ꢁ2
l
mol/ml and bactericidal effect at 1.44–2.92 ꢀ 10ꢁ2
lmol/
mol/
ml, followed by compound 4j with MIC 0.76–3.03 ꢀ 10ꢁ2
l
The synthesis of new compounds (Scheme 1) was carried out by
the reaction of the appropriate amine with chloroacetylchloride in
dry benzene under reflux for 3 h. Heterocyclization was performed
in the presence of ammonium thiocyanate refluxing in ethanol and
efficiently led to 2-(1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-ones
(3).21,22 After completion of the reaction, (usually 1 h), the 2-(thia-
zol-2-ylimino)thiazolidin-4-ones were afforded in excellent yields
and purity. The final compounds were obtained by refluxing the
previous intermediates with commercially available aldehydes
and anhydrous sodium acetate in glacial acetic acid. Reactions
were monitored by thin layer chromatography.
ml and MBC 1.56–6.06 ꢀ 10ꢁ2
lmol/ml, while 4o exhibited the
lowest inhibitory effect (0.73–23.3 ꢀ 10ꢁ2
lmol/ml) and bacterici-
dal activity (5.8–34.8 ꢀ 10ꢁ2
lmol/ml).
The obtained results support that the tested compounds exhib-
ited higher activity against all the screened bacteria, compared to
Streptomycin (MIC 4.3–25.8 ꢀ 10ꢁ2
lmol/ml, and MBC 8.6–
lmol/ml) and much better than Ampicillin (MIC
51.6 ꢀ 10ꢁ2
24.8–74.4 ꢀ 10ꢁ2
l
mol/ml, and MBC 37.2–124.0 ꢀ 10ꢁ2
lmol/ml).
Gram (+), Bacillus cereus was the most sensitive bacterial species
on compounds tested, while Staphylococcus aureus was the most
resistant one.
All new compounds were characterized by elemental and spec-
trophotometric analysis (IR and 1H NMR). The results are consis-
tent with the proposed structures and are in agreement with
previous findings.21
Taking under consideration the relationships between the
structure of the heterocyclic scaffold and the detected antibacterial
properties, it seems clear, that the introduction of the bulky bromo,
dimethylamino and chloro groups enhances the antibacterial
potential. It should also be mentioned that the presence of a meth-
oxy group is correlated with significant antibacterial activity (com-
pound 3).
In our previous papers21,22 have been already proposed a reac-
tion mechanism for the formation of the compounds under study.
2.2. Antimicrobial activity
Compounds 4h, 4j, 4s, 4i, 4k, 4e and 4c which are either
dimethylamino, or mono-chloro/bromo, mono-methoxy or
The designed compounds were evaluated for their in vitro anti-
microbial activity against seven bacterial and six fungal species
R
R
R
O
N
N
N
ClCOCH2Cl
N
NH4SCN
dry C6H6
EtOH, reflux
NHCOCH2Cl
S
2
NH2
S
1
N
H
S
S
reflux 3h
1h
3
R
R
O
N
OHC
N
N
S
S
CH3COOH
CH3COONa
reflux 2-4 h
H
R1
4
N/N
R
H
H
R1
N/N
R
R1
4a
3-OH
4k
4l
H
H
2,6-di-Cl
2,4-di-Cl
4b
3,5-di-OCH3, 4-
OH
4c
4d
4e
4f
4g
4h
4i
H
H
H
H
H
H
H
H
2-OCH3
2,5-di-OCH3
4-CH3
4m
4n
4o
4p
4q
4r
H
2,3-di-Cl
2-Cl
4-CH3
4-CH3
5-CH3
4-Ph
4-Ph
4-Ph
4-Ph
H
4-Cl
3-F
4-NO2
H
4-F
3-Br
2-Cl
4-Br
4s
4t
3-Cl
4j
N(CH3)2
4-Cl
4u
2-OH, 5-Br
Scheme 1. Reagents and conditions: (a) ClCOCH2Cl, dry C6H6, reflux,3 h; (b) NH4SCN, EtOH, reflux, 1 h; (c) RC6H4CHO, CH3COOH,CH3COONa, reflux, 2–4 h.

534
I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
Table 1
Antibacterial activity of unsubstituted/4-substituted 2-thiazolylimino-5-arylidene-4-thiazolidinones (MIC and MBC,
l
mol/ml ꢀ 10ꢁ2
)
Compounds
S. a.
B. c.
M. f
L. m.
Ps. aer.
S. typhi.
E. coli.
4b
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
11
5.5
11
11
22
3.15
3.15
23.2
29
11
22
3.15
6.30
5.8
11
11
27.5
33
27.5
3.15
6.30
23.2
34.8
2.47
3.3
38.5
3.15
6.30
29
27.5
3.15
6.30
11.5
23
4c
3.15
3.15
23.2
34.8
1.65
3.30
2.46
3.27
2.46
3.27
2.56
2.92
2.05
5.46
0.76
1.52
2.11
2.81
2.81
5.62
5.62
7.02
5.96
11.92
3.00
3.00
2.90
2.90
0.68
2.70
2.50
2.50
2.52
2.52
2.50
5.00
0.9
3.15
6.30
40.6
46.4
3.30
6.60
3.27
6.54
3.27
6.54
1.46
2.56
2.73
5.46
0.76
1.52
2.81
5.62
2.11
5.62
5.62
7.02
11.92
11.92
12.00
24.00
11.60
23.20
5.40
10.80
5.00
10.00
2.52
5.04
5.00
5.00
6.8
4d
23
40.6
1.65
2.47
2.46
3.27
3.27
6.54
1.46
2.56
2.73
5.46
3.03
6.06
2.11
2.81
2.81
7.02
2.81
5.62
5.96
11.92
3.00
12.00
2.90
11.60
2.70
10.80
5.00
10.00
2.52
5.04
5.00
10.00
1.7
4e
6.60
9.89
6.54
8.19
6.54
8.19
1.83
2.56
2.73
5.46
0.76
1.52
5.62
8.42
8.42
9.82
5.62
8.42
2.98
5.96
3.00
6.00
2.90
2.90
2.70
2.70
2.50
2.50
2.52
5.04
2.50
2.50
3.4
6.60
8.24
8.19
9.82
8.19
9.82
1.44
1.83
2.73
5.46
3.03
6.06
5.62
8.42
7.02
8.42
7.02
8.42
11.92
11.92
3.00
6.00
2.90
2.90
2.70
2.70
2.50
2.50
2.52
5.04
5.00
10.00
1.7
3.30
6.60
6.54
8.19
3.27
6.54
1.46
2.56
2.73
5.46
1.52
3.03
2.81
5.62
2.81
5.62
2.81
5.62
11.92
14.9
3.00
6.00
2.90
2.90
2.70
5.40
2.50
2.50
2.52
5.04
5.00
10.00
1.7
4f
3.27
6.54
3.27
6.54
0.73
1.44
1.37
2.73
3.03
6.06
2.81
5.62
5.62
7.02
7.02
8.42
5.96
11.92
12.00
24.00
11.60
23.20
5.40
10.80
5.00
10.00
2.52
5.04
5.00
10.00
3.4
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
6.8
3.4
6.8
3.4
3.4
3.4
13.7
37.20
49.20
17.20
34.40
Ampicilin
Streptomycin
24.80
37.20
17.20
34.40
24.80
37.20
4.30
8.60
24.80
37.20
8.60
17.20
37.20
74.40
25.80
51.60
74.40
124.00
17.20
34.40
24.80
49.20
17.20
34.40
S. a.—Staphylococcus aureus, B. c.—Bacillus cereus, M. f.—Micrococcus flavus, L. m.—Listeria monocytogenes, Ps. aer.—Pseudomonas aeruginosa, S. typhi.—Salmonella typhimurium,
E. coli.—Escherichia coli.
2,6-dichloro substituted derivatives, are presenting the most note-
worthy properties.
The results revealed very strong antifungal activity better than
commercial fungicides bifonazole (15- to 28-fold) and ketocona-
zole (15- to 250-fold) which were tested as positive standards (Ta-
ble 2). Minimal inhibitory concentration (MIC) of these compounds
followed by 4-Cl and 3-Cl derivatives. The last one is less potent
than the 5-(benzylidene)-2-(4-phenyl-1,3-thiazol-2-ylimino)-
1,3-thiazolidin-4-one It was also observed that the nature of
R-substituent affects the antifungal activity as well. Thus, replace-
ment of 4-phenyl- (compound 4t) with 4-methyl (4o) increased
activity 1.6-fold.
is observed at the range of 0.73–23.2 ꢀ 10ꢁ2
l
mol/ml and minimal
mol/ml. The anti-
It was found that the range of antibacterial activity of com-
pounds tested was wider than that of antifungal activity.
fungicidal concentration MFC 1.52–38.5 ꢀ 10ꢁ2
l
fungal potential could be presented as follows: 4j = 4o > 4r >
4n > 4i = 4t > 4h > 4q = 4s > 4p > 4c > 4l > 4m > 4f > 4e > 4u > 4k >
4g > 4b > 4d. The best activity was obtained by compound 4j with
2.3. Anti-inflammatory activity
MIC at the range of 0.76–1.52 ꢀ 10ꢁ2
l
mol/ml and MFC at the
2.3.1. In vivo inhibition of the carrageenin-induced edema
In acute toxicity experiments, the compounds examined in vivo
did not present toxic effects in ip doses up to 0.5 mmol/kg body
weight of the mouse. The in vivo anti-inflammatory effect of the
tested compounds was assessed by using the functional model of
carrageenin-induced mouse paw edema and is presented as the
percentage of inhibition of induction of edema at the right hind
paw (Table 3). Edema was measured by weight increase of the
right hind paw in comparison to the uninjected left paw.
range of 1.52 ꢀ 10ꢁ2
lmol/ml, while 4d showed the lowest anti-
fungal potential with MIC 0.73–23.3 ꢀ 10ꢁ2
lmol/ml and MFC at
5.8–34.8 ꢀ 10ꢁ2
lmol/ml. The most resistant fungal species was
Aspergillus flavus, while Trichoderma viride and Penicillium
ochrochloron were the most sensitive species.
Again the observed results support the significant positive
effect of bulky substituents to the antifungal potential.
It is worth to be mentioned that the position of chloro
substituent plays important role in antifungal activity. Thus, in
case of 5-(chlorobenzylidene)-2-(4-phenyl-1,3-thiazol-2-ylimi-
no)-1,3-thiazolidin-4-ones the most active appears to be 2-Cl,
Carrageenin-induced edema is a nonspecific inflammation that
is highly sensitive to nonsteroidal anti-inflammatory drugs (NSA-
IDs), and so carrageenin has been accepted as a suitable agent for

I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
535
Table 2
has been shown that inhibition of plant LOX activity by NSAIDs
is qualitatively similar to the inhibition of the rat mast cell LOX
and can be used as a simple qualitative screen for such activity.26
Perusal of IC50 values shows that compound 4d is the most ac-
tive within the set, followed by compounds 4t, 4c, 4o, 4n = 4p (Ta-
ble 3). The comparison of 4-Cl derivatives (4o) and (4t) revealed
that compound 4t demonstrates highly significant inhibition com-
pared to the corresponding 4-CH3 substituted derivative (4o).
Antifungal activity of unsubstituted/4-substituted 2-thiazolylimino-5-arylidene-4-
thiazolidinones (MIC and MFC,
l
lmol/ml ꢀ 10-2
)
Compounds
P. f.
P. o.
T. v.
A. fum.
A. n.
A. fl.
4b
4c
4d
4e
4f
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
5.5
11
1.58
3.15
0.73
5.8
1.38
2.75
1.58
3.15
5.8
11
22
1.58
3.15
5.8
22
33
22
11
22
2.36
3.15
29
38.5
1.58
3.15
23.2
29
1.58
3.15
23.3
34.8
4.94
6.60
3.27
4.91
4.91
6.54
1.80
2.56
1.37
2.73
1.52
1.52
5.62
7.02
4.21
5.62
5.62
7.02
1.79
2.09
1.52
1.52
1.58
3.15
1.90
2.52
1.76
2.34
1.90
2.52
1.37
2.73
1.7
11.6
3.30
6.60
3.27
6.54
3.27
6.54
1.80
2.20
1.37
2.73
0.76
1.52
2.81
5.62
4.21
5.62
4.21
5.62
1.49
2.09
0.76
1.52
1.58
3.15
1.90
2.52
1.17
2.34
1.90
2.52
1.37
2.73
1.7
34.8
3.30
4.94
3.27
6.54
3.27
6.54
1.80
2.20
1.37
2.73
0.76
1.52
2.11
2.81
2.81
4.21
2.81
5.62
1.79
2.09
0.76
1.52
1.58
3.15
1.26
2.52
1.17
2.34
1.26
2.52
1.37
2.73
0.85
1.7
34.8
4.94
6.60
3.27
6.54
6.54
8.19
2.20
2.56
1.37
2.73
1.52
1.52
5.62
7.02
4.21
5.62
4.21
5.62
2.09
2.09
1.52
1.52
1.58
3.15
1.90
2.52
1.76
2.34
1.90
2.52
1.37
2.73
3.4
2.3.3. COX-1/COX-2 inhibition
4.94
6.60
3.27
6.54
3.27
6.54
1.80
2.20
2.05
2.73
0.76
1.52
2.81
7.02
2.81
5.62
2.81
5.62
1.79
2.09
0.76
1.52
1.58
3.15
1.90
2.52
1.76
2.34
1.90
2.52
2.05
2.73
1.7
4.94
6.60
3.27
6.54
3.27
6.54
1.80
2.56
1.37
2.73
1.52
1.52
5.62
7.02
4.21
5.62
4.21
5.62
1.49
2.09
1.52
1.52
1.58
3.15
1.90
2.52
1.76
2.34
1.90
2.52
1.37
2.73
3.4
Cycloxygenase inhibitory action was measured using ovine re-
combinant COX-1 and and human recombinant COX-2 isoenzymes
included in ‘COX inhibitor screening assay’ kit purchased by
Cayman. It was found that all tested compounds exhibited inhibi-
tory activity ranging from 50% to 86%. The best inhibition was
4g
4h
4i
observed for compound 4m (100%, IC50 10
lM) followed by
compound 4a (98%, IC50 16 M). It seems that the presence of a
l
chloro substituent (4k–4o, 4s, 4t) in general is favourable for
COX inhibitory activity. It should be mentioned that two chloro
atoms in the molecule leads to more active COX-1 inhibitors
(97–100%) in comparison to mono chloro substitution (65–97%).
The replacement of 3-Cl and 4-Cl with a fluoro substituent de-
creased activity two-folds, while in case of bromo derivatives
inhibition seems to be dependent on position of the bromine atom
in the molecule. Thus, 4-Br derivative exhibited moderate activity
(67%) while 3-Br was almost inactive (2%) (Table 3). Good inhibi-
tory activity was exhibited by 5-(4-(chlorobenzylidene)-2-(4-
methyl-1,3-thiazol-2-ylimino)-1,3-thiazolidin-4-one (4e).
On the other hand it is obvious that the presence of free OH
group in C3 of benzene ring is also beneficial for COX-1 inhibition.
The insertion of methoxy group in 3,5 position led to dramatic de-
crease of inhibitory activity. The role of lipophilicity is not well
defined.
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
All compounds showed no or low (0–47%) COX-2 inhibition
when they were added to the assay mixture in a concentration of
4t
200
lM. The results shown in Table 3 were obtained at an
4u
Bif
Ket
arachidonic acid substrate concentration of 0.1
lM. Increase of
3.4
3.4
3.4
6.8
6.8
64.00
80.00
38.00
95.00
48.00
64.00
380.00
380.00
64.00
80.00
475.00
570.00
48.00
64.00
38.00
95.00
48.00
64.00
38.00
95.00
48.00
64.00
285.00
380.00
arachidonic acid concentration led to loss of activity, showing that
the compounds are competitive inhibitors of the enzyme.
5-(2-Methoxybenzylidene)-2-(4-phenyl-1,3-thiazol-2-ylimino)-
1,3-thiazolidin-4-ones (4c) exhibited the best COX-2 inhibition
(47%). It is only 1.4-fold less active than naproxene. Introduction
of the second methoxy group led to compound 4d which is com-
pletely inactive as COX-2 inhibitor. Inhibition was also exhibited
by compound 4t (34%) with 4-Cl substitution. The results show that
lipophilicity does not play role in COX-2 inhibition.
P. f.—Penicillium funiculosum, P. o.—Penicillium ochrochloron, T. v.—Trichoderma viride,
A. fum.—Aspergillus fumigatus, A. n.—Aspergillus niger, A. fl.—Aspergillus flatus.
the study of new compounds with anti-inflammatory activity. As
shown in Table 3, the majority of the investigated compounds in-
duced protection against carrageenin-induced paw edema. The
protection ranged up to 67.3%, while the reference drug, indometh-
acin exhibited 47% protection at an equivalent dose. Compound 4j
is the most potent followed by compounds 4q, 4k and 4p. Among
the 3-Cl and 4-Cl substituted derivatives, compounds 4s and 4t and
the unsubstituted 4q, most potent seems to be compound 4q. It
3. Conclusion
This study has demonstrated that 5-arylidene-2-(1,3-thiazol-2-
ylimino)-1,3-thiazolidin-4-ones bearing a variety of substituents in
the aromatic part combine anti-inflammatory, antibacterial and
antifungal activities. All tested compounds exhibited good ability
to inhibit bacteria and fungi, higher than commercial antimicrobial
agents used as reference drugs. It is worth to notice that these
compounds are more potent/selective against fungi.
seems that low
anti-inflammatory activity; for example, for compounds 4j and
4i, 4-N(CH3)2 >4-Br ( -N(CH3)2 = 0.18; -Br 0.86. Between the
p values of R1 are correlated with higher
p
p
two 4-CH3 substituted analogues (4n and 4o) no differences are
Some of our derivatives proved to be potent in vitro inhibitors
of soybean lipoxygenase and COX-1 enzyme. Compound 4d is the
most potent LOX inhibitor, whereas compound 4j presents the
highest anti-inflammatory activity and 50% COX-1 inhibition. Com-
pound 4m (IC50 10 lM) is the most potent COX-1 inhibitor. The
influence of lipophilicity is not well documented.
observed. Both compounds seem to be equipotent.
Based on very preliminary results, it seems that molar
hydrophilicity (low lipophilicity values, ClogP) partly affects the
anti-inflammatory activity of the sub-group in which R = H. On
the contrary, for the sub-group where R is phenyl, higher lipophil-
icity is correlated with potent anti-inflammatory activity in vivo.
However, further investigation is needed in order to gain insight
into the mechanism of action of the examined compounds. Some of
the synthesized compounds might constitute initial leads for the
design of new more potent multi-target therapeutic agents.
2.3.2. In vitro soybean lipoxygenase LOX inhibition
Compounds were further evaluated for inhibition of soybean
lipoxygenase LOX by the UV absorbance based enzyme assay. It

536
I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
Table 3
Anti-inflammatory and COX/LOX inhibitory activity of synthesized compounds
Compound
CPE (%)a
COX-1 (%)
COX-1 (IC50
lM)
COX-2 (%)
LOX (%)
LOX (IC50
lM)
ClogP
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
33.7
39.0
50.0 2.2
26.0 1.1
47.0
56.57
65.6
42.7
42.2 1.3
67.3 4.5
58.5
9.9
45.0
43.0 3.4
42.0 3.2
55.4 3.1
59.3 2.1
51.0 1.8
27.3 1.0
54.4
47.0 1.1
Nt
Nt
98.0
23.0
86.0
85.0
99.0
42.0
44.0
2.0
67.0
50.0
100.0
98.0
100.0
97.0
74.0
68.0
67.0
65.0
78.0
73.0
Nt
16.0
—
—
—
51.0
—
—
—
—
—
78.0
32.0
10.0
—
—
—
—
—
—
65
—
18.0
0
47.0
0
Nt
16.0
11.0
7.0
0
35.0
11.0
>150
>150
63.0
52.0
92.0
90.0
>150
>150
66.0
95.0
137
122.0
143.0
79.0
76.0
79.0
66.0
88.0
54.0
0.393
1.530
2.490
1.927
1.571
1.571
2.291
2.291
2.260
2.854
2.854
2.734
2.820
2.820
67.0
72.0
35.0
34.0
0
22.0
Nt
Nt
0
0
Nt
0
43.0
54.0
41.0
4q
4s
4t
4u
3.700
4.420
4.420
0
34.0
Nt
Nt
65.0
Nt
Nt
Indom/cine
Naproxene
NDGA
Nt
Nt
32
46.0
Nt
49.0
—
a
Inhibitory activity on carrageenan-induced rat paw oedema. The results are expressed as mean (n = 6–10) percentage reduction of the difference in weight between the
carrageenan injected and uninjected paws following a 0.01 mmol/kg intraperitoneal injection of the test compound.
ppm, DMSO-d6, 300 MHz): 6.88–6.91 (m, 1H, ArH), 7.04–7.10 (m,
4. Experimental
2H, ArH), 7.33–7.38 (m, 1H, ArH), 7.49–7.62 (m, 2H, thiazole),
7.73 (s, 1H, @CH). Anal. Calcd for C13H9N3O2S2 (303.01): C, 51.47;
H, 2.99; N, 13.85. Found: C, 51.50; H, 3.01; N, 13.83.
4.1. Chemistry—general aspects
Melting points °C were determined with a MELTEMP II capillary
apparatus (LAB Devices, Holliston, MA, USA) without correction.
Elemental analyses were performed on Perkin–Elmer 2400 CHN
elemental analyzer for all compounds synthesized and were within
0.4% of theoretical values. IR spectra were recorded, in Nujol, on
Perkin Elmer Spectrum BX and DR-8001 Shimadzu. Wave numbers
4.1.1.2. 5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-(1,3-thia-
zol-2-ylimino)-1,3-thiazolidin-4-one (4b).
201–202 °C. IR (Nujol): 3089 (NH), 1702 (C@O), 1591 (C@N)
cmꢁ1 1H NMR (d ppm, DMSO-d6, 300 MHz): 3.84 (s, 6H, OCH3),
Yield: 11%, mp
.
6.97(s, 2H, ArH 20 and 60), 7.38–7.45 (m, 1H, thiazole 50), 7.58–
7.65 (m, 2H, thiazole 40, @CH). Anal. Calcd for C15H13N3O4S2
(363.03): C, 49.57; H, 3.61; N, 11.56. Found: C, 49.55; H, 3.59; N,
11.54.
in the IR spectra are given in cmꢁ1 1H NMR spectra of the newly
.
synthesized compounds, in DMSO-d6 solutions, were recorded on
a Bruker AC 300 instrument (Bruker, Karlsruhe, Germany) at
298 K. Chemical shifts are reported as d (ppm) relative to TMS as
internal standard. Coupling constants J are expressed in Hertz
(Center of Instrumental Analysis of the University of Thessaloniki).
The reactions were monitored by TLC on F254 silica-gel pre-
coated sheets (Merck, Darmstadt, Germany) and the purified com-
pounds each showed a single spot.
Solvents, unless otherwise specified were of analytical reagent
grade or of the highest quality commercially available. Synthetic
starting materials, reagents and solvents were purchased from Al-
drich Chemie (Steinheim Germany).
4.1.1.3.
1,3-thiazolidin-4-one (4c).
(Nujol): 3110 (NH), 1690 (C@O), 1597 (C@N). cmꢁ1
5-(2-Methoxybenzylidene)-2-(1,3-thiazol-2-ylimino)-
Yield: 76%, mp 234–235 °C. IR
1H NMR (d
.
ppm, DMSO-d6, 300 MHz): 3.90 (s, 3H, OCH3), 7.15–7.18 (m, 2H,
ArH), 7.46–7.53 (m, 3H, ArH, thiazole), 7.69–7.70 (m, 1H, ArH),
7.94 (s, 1H, @CH). Anal. Calcd for C14H11N3O2S2 (317.39): C,
52.98; H, 3.49; N, 13.24. Found: C, 52.99; H, 3.52; N, 13.26.
4.1.1.4. 5-(2,5-Dimethoxybenzylidene)-2-(1,3-thiazol-2-ylimi-
no)-1,3-thiazolidin-4-one (4d).
Yield: 84%, mp 195–197 °C.
4.1.1. General procedure for synthesis of 5-arylidene-2-(4-
substituted/unsubstituted-1,3-thiazol-2-ylimino)-1,3-
thiazolidin-4-ones (4a–4u)
A well-stirred solution of 0.8 g of 2-(thiazol-2-ylimino)thiazoli-
din-4-one (4 mmol) in 35 mL of acetic acid was buffered with so-
dium acetate (8 mmol) and added with the appropriate
arylaldehyde (6 mmol). The solution was refluxed for 4 h and then
poured into ice-cold water. The precipitate was filtered and
washed with water and the resulting crude product was purified
by recrystallisation from dioxane.
1H NMR (d ppm, DMSO-d6, 300 MHz): 3.78 (s, 3H, 5-OMe), 3.85
(s, 3H, 2-OMe), 7.04–7.1 (m, 3H, ArH), 7.47 (d, J = 3.3 Hz, 1H, thia-
zole 50), 7.7 (d, J = 3.6 Hz, 1H, thiazole 40), 7.87 (s, 1H, @CH). Anal.
Calcd for C15H13N3O3S2 (347.42): C, 51.86; H, 3.77; N, 12.10. Found:
C, 51.82; H, 3.84; N, 12. 15.
4.1.1.5. 5-(4-Methylbenzylidene)-2-(1,3-thiazol-2-ylimino)-1,3-
thiazolidin-4-one (4e).
Yield: 13%, mp 211–213 °C. IR (Nujol):
3195 (NH), 1717 (C@O), 1584 (C@N) cmꢁ1. 1H NMR (d ppm, DMSO-
d6, 300 MHz): 2.37 (s, 3H, Ar-CH3), 7.30–7.39 (m, 2H, ArH 30 and 50),
7.52–7.55 (m, 3H, ArH 20 and 60, thiazole 50), 7.68 (s, 1H thiazole 40),
7.72 (s, 1H, @CH). Anal. Calcd for C14H11N3OS2 (301.03): C, 55.79; H,
3.68; N, 13.94. Found: C, 55.82; H, 3.71; N, 13. 97.
4.1.1.1.
1,3-thiazolidin-4-one (4a).
(Nujol): 3084 (NH), 1702 (C@O), 1594 (C@N) cmꢁ1
5-(3-Hydroxybenzylidene)-2-(1,3-thiazol-2-ylimino)-
Yield: 12%, mp 279–280 °C. IR
1H NMR (d
.

I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
537
4.1.1.6. 5-(3-Fluorobenzylidene)-2-(1,3-thiazol-2-ylimino)-1,3-
thiazolidin-4-one (4f). Yield: 34%, mp 217–219 °C. IR (Nu-
jol): 3091 (NH), 1720 (C@O), 1596 (C@N) cmꢁ1 1H NMR (d ppm,
thiazole). Anal. Calcd for C14H10ClN3OS2 (335.83): C, 50.07; H, 3.00;
N, 10.56. Found: C, 50.05; H, 2.98; N, 10.54.
.
DMSO-d6, 300 MHz): 7.30–7.32 (m, 1H, ArH), 7.47–7.59 (m, 2H thi-
azole), 7.61–7.66 (m, 3H, ArH), 7.71 (s, 1H, @CH). Anal. Calcd for
4.1.1.15.
5-(4-Chlorobenzylidene)-2-(4-methyl-1,3-thiazol-2-
ylimino)-1,3-thiazolidin-4-one (4o).
Yield: 77%, mp 270–
C13H8FN3OS2 (305.01): C, 51.13; H, 2.64; N, 13.76. Found: C,
271 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 2.37 (s, 3H, CH3),
7.64–7.70 (m, 6H, ArH, thiazole, @CH). Anal. Calcd for
51.16; H, 2.67; N, 13.79.
C14H10ClN3OS2 (335,83): C, 50.07; H, 3.00; N, 12.51. Found: C,
4.1.1.7.
5-(4-Fluorobenzylidene)-2-(1,3-thiazol-2-ylimino)-
50.10; H, 3.03; N, 12.54.
1,3thiazolidin-4-one (4g).
Yield: 27%, mp 225–227 °C. IR
(Nujol): 3091 (NH), 1720 (C@O), 1596 (C@N) cmꢁ1
.
1H NMR (d
4.1.1.16. 5-(4-Nitrobenzylidene)-2-2-(5-methy-1,3-thiazol-2-yli-
ppm, DMSO-d6, 300 MHz): 7.39–7.58 (m, 4H, ArH), 7.69–7.73 (m,
3H, thiazole, @CH). Anal. Calcd for C13H8FN3OS2 (305.01): C,
51.13; H, 2.64; N, 13.76. Found: C, 51.15; H, 2.66; N, 13.78.
mino)-1,3-thiazolidin-4-one (4p).
Yield: 52%, mp 301–
303 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 2.37 (s, 3H, CH3),
7.64–7.69 (m, 6H, ArH, thiazole, @CH). Anal. Calcd for
C14H10N4O3S2 (346.38): C, 48.54; H, 2.91; N, 16.17. Found: C,
4.1.1.8. 5-(3-Bromobenzylidene)-2-(1,3-thiazol-2-ylimino)-1,3-
48.50; H, 3.03; N, 16.24.
thiazolidin-4-one (4h).
Yield: 38%, mp 209–210 °C. IR (Nu-
jol): 3098 (NH), 1709 (C@O), 1596 (C@N) cmꢁ1
.
1H NMR (d ppm,
4.1.1.17. 5-Benzylidene-2-(4-phenyl-1,3-thiazol-2-ylimino)-1,3-
DMSO-d6, 300 MHz): 7.51–7.57 (m, 2H, thiazole), 7.74 (d,
J = 7.5 Hz, 2H, ArH), 7.71 (s, 1H, ArH 20), 7.62–7.67 (m, 1H, ArH),
7.87 (s, 1H, @CH). Anal. Calcd C13H8BrN3OS2 (364.93): C, 42.63;
H, 2.20; N, 11.47. Found: C, 42.61; H, 2.18; N, 11.45.
thiazolidin-4-one (4q).
(d ppm, DMSO-d6, 300 MHz): 7.39–7.75 (m, 9H, ArH, thiazole),
7.89 (s, 1H, @CH), 8.01 (d, J = 7.5 Hz, 2H, ArH). Anal. Calcd for
Yield: 84%, mp 285–287 °C. 1H NMR
C19H13N3OS2 (363.46): C, 62.79; H, 3.61; N, 11.56. Found: C,
62.84; H, 3.71; N, 11.60
4.1.1.9. 5-(4-Bromobenzylidene)-2-(1,3-thiazol-2-ylimino)-1,3-
thiazolidin-4-one (4i).
Yield: 80, mp 268–269 °C. 1H NMR
4.1.1.18. 5-(2-Chlorobenzylidene)-2-(4-phenyl-1,3-thiazol-2-yli-
(d ppm, DMSO-d6, 300 MHz): 7.51 (d, J = 3 Hz, 1H, thiazole 50),
7.58 (d, J = 9 Hz, 2H, ArH 30 and 50), 7.68–7.72 (m, 1H, thiazole
40), 7.73 (s, 1H, @CH), 7.78 (d, J = 9 Hz, 2H, ArH 20 and 60). Anal.
Calcd for C13H8BrN3OS2 (364.93): C, 42.63; H, 2.20; N, 11.47.
Found: C, 42.67; H, 2.23; N, 11.50.
mino)-1,3-thiazolidin-4-one (4r).
Yield: 64%, mp 256–
258 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 7.34–7.69 (m, 6H,
ArH), 7.80 (d, 1H, ArH), 7.87 (s, 1H, @CH), 7.93–7.97 (m, 3H, thia-
zole, ArH). Anal. Calcd for C19H12ClN3OS2 (397.90): C, 57.35; H,
3.04; N, 10.56. Found: C, 57.42; H, 3.12; N, 10.49.
4.1.1.10.
5-(4-(Dimethylamino)benzylidene)-2-(1,3-thiazol-2-
Yield: 69%, mp 176–
4.1.1.19.
5-(3-Chlorobenzylidene)-2-(4-phenyl-1,3-lthiazol-2-
ylimino-1,3-thiazolidin-4-one (4j).
ylimino)-1,3-thiazolidin-4-one (4s).
Yield: 76%, mp 304–
177 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 3.03 (s, 6H,
N(CH3)2), 6.86 (d, J = 7.2 Hz, 2H, ArH 30 and 50), 7.44–7.70 (m, 5H,
ArH 20 and 60, thiazole, @CH). Anal. Calcd for C15H14N4OS2
(330.43): C, 54.52; H, 4.27; N, 16.96. Found: C, 54.62; H, 4.29; N,
16.87.
305 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 7.36–7.72 (m, 7H,
ArH), 7.78 (s, 1H, @CH), 7.94 (s, 1H, thiazole), 8.01 (d, 2H, ArH).
Anal. Calcd for C19H12ClN3OS2 (3971): C, 57.35; H, 3.04; N, 10.56.
Found: C, 57.38; H, 3.09; N, 10.59.
4.1.1.20. 5-(4-Chlorobenzylidene)-2-(4-phenyl-1,3-thiazol-2-yli-
4.1.1.11.
no)1,3-thiazolidin-4-one (4k).
IR (Nujol): 3079 (NH), 1693 (C@O), 1590 (C@N) cmꢁ1
ppm, DMSO-d6, 300 MHz): 7.47–7.54 (m, 2H, thiazole), 7.62–7.64
(m, 4H, ArH and @CH). Anal. Calcd for C13H7Cl2N3OS2 (354.94): C,
43.83; H, 1.98; N, 11.80. Found: C, 43.80; H, 1.95; N, 11.95.
5-(2,6-Dichlorobenzylidene)-2-(1,3-thiazol-2-ylimi-
Yield: 74%, mp 255–257 °C.
1H NMR (d
mino)-1,3-thiazolidin-4-one (4t).
Yield: 77%, mp 247–8 °C.
IR (Nujol): 3200 (NH), 1690 (C@O), 1650 (C@N) cmꢁ1
.
1H NMR (d
.
ppm, DMSO-d6, 300 MHz): 7.48–7.77 (m, 8H, ArH, thiazole),
7.93–8.02 (m, 3H, @CH, ArH). Anal. Calcd for C19H12ClN3OS2
(397.91): C, 57.35; H, 3.04; N, 10.56. Found: C, 57.40; H, 3.10; N,
10.60.
4.1.1.12. 5-(2,4-Dichlorobenzylidene)-2-(1,3-thiazol-2-ylimino-
4.1.1.21. 5-(2-Hydroxy-5-bromobenzylidene)-2-(1,3-thiazol-2-
1,3-thiazolidin-4-one (4l).
Yield: 75%, mp 259–261 °C. IR
ylimino)-1,3-thiazolidin-4-one (4u).
Yield: 79%, mp 185–
(Nujol): 3081 (NH), 1696 (C@O), 1593 (C@N) cmꢁ1
.
1H NMR (d
186 °C. IR (Nujol): 3200 (NH), 1690 (C@O), 1650 (C@N) cmꢁ1
.
1H
ppm, DMSO-d6, 300 MHz): 7.48–7.49 (m, 1H, thiazole 50), 7.50–
7.68 (m, 3H, thiazole 40, ArH), 7.70–7.79 (m, 2H, ArH, @CH). Anal.
Calcd for C13H7Cl2N3OS2 (354.94): C, 43.83; H, 1.98; N, 11.80.
Found: C, 43.82; H, 1.97; N, 11.79.
NMR (d ppm, DMSO-d6, 300 MHz): 6.98 (d, J = 8.7 Hz, 1H, ArH),
7.59 (d, J = 3.6 Hz, 1H, thiazole 50), 7.64 (d, J = 2.7 Hz, 1H, ArH),
7.67 (d, J = 2.4 Hz, 1H, thiazole 40), 7.71 (d, J = 2.7 Hz, 2H, @CH,
ArH), 10.21 (s, 1H, OH). Anal. Calcd for C13H8BrN3O2S2 (382.25):
C, 40.85; H, 2.11; N, 10.99. Found: C, 40.87; H, 2.14;N, 11.015.
4.1.1.13. 5-(2,3-Dichlorobenzylidene)-2-(1,3-thiazol-2-ylimino)-
1,3-thiazolidin-4-one (4m).
Yield: 83%, mp 264–265 °C. IR
(Nujol): 3084 (NH), 1671 (C@O), 1595 (C@N) cmꢁ1
.
1H NMR (d
4.2. Antibacterial activity
ppm, DMSO-d6, 300 MHz): 7.49 (d, J = 3 Hz, 1H, thiazole 50), 7.59–
7.69 (m, 3H, thiazole 40, ArH), 7.75 (d, J = 6 Hz, 1H, ArH), 7.83 (s,
1H, @CH). Anal. Calcd for C13H7Cl2N3OS2 (354.94): C, 43.83; H,
1.98; N, 11.80. Found: C, 43.82; H, 1.97; N, 11.77.
The following Gram-negative bacteria were used: Escherichia
coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmo-
nella typhimurium (ATCC 13311), and the following Gram-positive
bacteria: Listeria monocytogenes (NCTC 7973), Bacillus cereus (clin-
ical isolate), Micrococcus flavus (ATCC 10240), and Staphylococcus
aureus (ATCC 6538). The organisms were obtained from the Myco-
logical Laboratory, Department of Plant Physiology, Institute for
4.1.1.14.
5-(2-Chlorobenzylidene)-2-(4-methyl-1,3-thiazol-2-
ylimino)-1,3-thiazolidin-4-one (4n).
Yield: 65%, mp 241–
243 °C. 1H NMR (d ppm, DMSO-d6, 300 MHz): 2.26 (s, 3H, thiazole
4-CH3), 7.01 (d, 1H, ArH), 7.48–7.69 (m, 4H, ArH, @CH), 7.84 (s, 1H,
´
Biological Research ‘Siniša Stankovic’, Belgrade, Serbia.

538
I. Apostolidis et al. / Bioorg. Med. Chem. 21 (2013) 532–539
The antibacterial assay was carried out by microdilution meth-
3000 lg/ml). All experiments were performed in duplicate and
od27–29 in order to determine the antibacterial activity of com-
pounds tested against the human pathogenic bacteria.
The bacterial suspensions were adjusted with sterile saline to a
concentration of 1.0 ꢀ 105 cfu/ml. The inocula were prepared daily
and stored at +4 °C until use. Dilutions of the inocula were cultured
on solid medium to verify the absence of contamination and to
check the validity of the inoculum.
repeated three times.
4.4. Biological assays: in vitro experiments
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 average values.
4.2.1. Microdilution test
4.4.1. Soybean lipoxygenase inhibition study in vitro31
The minimum inhibitory and bactericidal concentrations (MICs
and MBCs) were determined using 96-well microtitre plates. The
bacterial suspension was adjusted with sterile saline to a concen-
tration of 1.0 ꢀ 105 cfu/ml. Compounds to be investigated were
Lipoxygenase inhibition was evaluated as reported previously.9
The tested compounds, dissolved in DMSO, were added to the reac-
tion mixture at several concentrations and were preincubated for
4 min at 28 °C with soybean lipoxygenase at a concentration of
7 ꢀ 10ꢁ7 w/v. IC50 values were determined. Enzyme reaction was
initiated by the addition of sodium linolate at a final concentration
dissolved in broth LB medium (100 ll) with bacterial inoculum
(1.0 ꢀ 104 cfu per well) to achieve the wanted concentrations
(1 mg/ml). The microplates were incubated for 24 h at 48 °C. The
lowest concentrations without visible growth (at the binocular
microscope) were defined as concentrations that completely inhib-
ited bacterial growth (MICs). The compounds investigated were
dissolved in 5% DMSO (1 mg/ml) and added in LB medium with
inoculum. The MBCs were determined by serial sub-cultivation of
of 100 lM. The conversion of sodium linoleate to 13-hydroperoxy-
linoleic acid was measured at 234 nm. Nordihydroguaretic acid, an
appropriate standard inhibitor, was used as positive control (inhi-
bition) 94.4% at 100 lM.
4.4.2. COX inhibitor screening assay32
2
l
l into microtitre plates containing 100
l
l of broth per well
The COX-1 and COX-2 activities of the compounds were mea-
sured using ovine COX-1 and human recombinant COX-2 enzymes
included in the ‘COX inhibitor screening assay’ kit provided by Cay-
man (Cayman Chemical Co., Ann Arbor, MI). The assay directly
measures PGF2a produced by SnCl2 reduction of COX-derived
PGH2. The prostanoids production was quantified via enzyme
immunoassay using a broadly specific antibody that binds to all
the major prostaglandin compounds. COX-1/COX-2 inhibitory
activity was tested at an arachidonic acid concentration of
and further incubation for 72 h. The lowest concentration with
no visible growth was defined as the MBC, indicating 99.5% killing
of the original inoculum. The optical density of each well was
measured at a wavelength of 655 nm by Microplate manager 4.0
(Bio-Rad Laboratories) and compared with a blank and the positive
control. Streptomycin and ampicillin were used as a positive con-
trol (1 mg/ml). All experiments were performed in duplicate and
repeated three times.
0.1 lM which is much lower from the saturating concentration.
4.3. Antifungal activity
IC50 values were calculated for the most active compounds. Na-
proxen was used as positive control.
For the antifungal bioassays, eight fungi were used: Aspergillus
flavus (ATCC 9643), Aspergillus fumigatus (plant isolate), Aspergillus
niger (ATCC 6275), Penicillium funiculosum (ATCC 36839), Penicil-
lium ochrochloron (ATCC 9112) and Trichoderma viride (IAM
5061). The organisms were obtained from the Mycological Labora-
tory, Department of Plant Physiology, Institute for Biological Re-
4.4.3. In vivo experiments: inhibition of the carrageenin-
induced edema
Edema was induced in the right hind paw of mice (AKR) by the
intradermal injection of 0.05 mL of 2% carrageenin in water. Both
sexes were used, but pregnant females were excluded. Each group
was composed of 6–10 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 libi-
tum prior to experimentation, but they were fasted during the
experimental period. 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 and were given intraperitoneally
simultaneously with the carrageenin injection. The mice were
euthanized 3.5 h after the carrageenin injection. The difference be-
tween the weight of the injected and uninjected paws was calcu-
lated for each animal. It was compared with that in control
animals (treated with water) and expressed as a percent inhibition
of the edema CPE% values (Table 3). Each experiment was per-
formed in duplicate, and the standard deviation was less than 10%.
´
search ‘‘Siniša Stankovic’’, Belgrade, Serbia.
The micromycetes were maintained on malt agar and the cul-
tures stored at 4 °C and sub-cultured once a month30 In order to
investigate the antifungal activity of the extracts, a modified mic-
rodilution technique was used.27–29 The fungal spores were
washed from the surface of agar plates with sterile 0.85% saline
containing 0.1% Tween 80 (v/v). The spore suspension was adjusted
with sterile saline to a concentration of approximately 1.0 ꢀ 105 in
a final volume of 100 ll per well. The inocula were stored at 4 °C
for further use. Dilutions of the inocula were cultured on solid malt
agar to verify the absence of contamination and to check the valid-
ity of the inoculum.
Minimum inhibitory concentration (MIC) determinations were
performed by a serial dilution technique using 96-well microtiter
plates. The compounds investigated were dissolved in 5% DMSO
(1 mg/ml) and added in broth Malt medium with inoculum. The
microplates were incubated for 72 h at 28 °C, respectively. The
lowest concentrations without visible growth (at the binocular
microscope) were defined as MICs.
Acknowledgments
We thank Dr. A. Leo and Biobyte Corp., Claremont, California, for
free access to the C-QSAR program.
The fungicidal concentrations (MFCs) were determined by se-
rial subcultivation of a 2
ll into microtiter plates containing
References and notes
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l
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Commemorative Issue in Honor of Prof. Henk van der Plas on the occasion of
his 80th anniversary.