5-Arylidene-4-thiazolidinone derivatives active as antidegenerative agents on human chondrocyte cultures

Article abstract of DOI:10.2174/157340613804488378

5-Arylidene-2-oxo-4-thiazolidinones and 2-phenylimino analogues were evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1β and for their inhibitory capability against matrix metalloproteinase- 13. Our results indicated that 5-arylidene-4-thiazolidinone derivatives 1-9 exhibit antidegenerative activity and could block multiple cartilage destruction during the osteoarthritic process. Out of the selected compounds, (5-arylidene- 2,4-dioxothiazolidin-3-yl)acetic acids 7-9 showed significant effectiveness in reducing NO release and restoring normal levels of GAGs in chondrocytes treated with IL-1β. Moreover, benzoic acids 1, 5 and 6 proved to be effective MMP-13 inhibitors and were able to restore normal levels of GAGs.

Full text of DOI:10.2174/157340613804488378

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84
Medicinal Chemistry, 2013, 9, 84-90
5-Arylidene-4-Thiazolidinone Derivatives Active as Antidegenerative
Agents on Human Chondrocyte Cultures
Annamaria Panico1, Rosanna Maccari2, Venera Cardile3, Lucia Crascì1, Simone Ronsisvalle1 and
Rosaria Ottanà2,*
1Dipartimento di Scienze del Farmaco, Faculty of Pharmacy, University of Catania, V.le A. Doria 6, 95125 Catania,
Italy
2Dipartimento Farmaco-chimico, Faculty of Pharmacy, University of Messina, Polo Universitario dell’Annunziata,
98168 Messina, Italy
3Dipartimento di Scienze Fisiologiche, Faculty of Science, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
Abstract: 5-Arylidene-2-oxo-4-thiazolidinones and 2-phenylimino analogues were evaluated for their antidegenerative
activity on human chondrocyte cultures stimulated by IL-1ꢀ and for their inhibitory capability against matrix metallopro-
teinase-13. Our results indicated that 5-arylidene-4-thiazolidinone derivatives 1-9 exhibit antidegenerative activity and
could block multiple cartilage destruction during the osteoarthritic process. Out of the selected compounds, (5-arylidene-
2,4-dioxothiazolidin-3-yl)acetic acids 7-9 showed significant effectiveness in reducing NO release and restoring normal
levels of GAGs in chondrocytes treated with IL-1ꢀ. Moreover, benzoic acids 1, 5 and 6 proved to be effective MMP-13
inhibitors and were able to restore normal levels of GAGs.
Keywords: Antidegenerative activity, Anti-inflammatory activity, 5-Arylidene-2-phenylimino-4-thiazolidinones, 5-Arylidene-
4-thiazolidinones, Glycosaminoglycans, Human chondrocytes, Matrix metalloproteinase-13, Nitric oxide.
INTRODUCTION
tory cytokines (e.g., IL-1ꢀ and TNF-ꢀ), free radicals (ROS
and NO) and matrix metalloproteinases (MMPs) [2,3].
Osteoarthritis (OA) is the most common form of joint
disease that evolves from a local inflammatory disease to a
chronic process with distinct inflammatory and destructive
components. The progressive damage of the articular carti-
laginous tissue represents a central moment of the pathoge-
netic course, in which depletion of aggrecan, disregulated
synthesis of matrix components such as proteoglycans and
collagen play a fundamental role [1].
Cytokines interfere in the ECM turnover, accelerate the
degradation of cartilage matrix, induce high levels of proin-
flammatory mediators, such as prostaglandins E2 (PGE2) and
nitric oxide (NO), and inhibit the synthesis of both collagen
and PGs. In particular, interleukin-1ꢀ (IL-1ꢀ) is a pivotal
driving force in inducing and sustaining cartilage damage
also by means of the loss of GAGs. Moreover, it induces an
increased expression of the nuclear factor kB (NF-kB),
which in turn promotes the transcription of different genes
codifying NO synthase, cyclooxygenase (COX), intracellular
adhesion molecule-1 (ICAM-1) [4,5].
Cartilage is composed by chondrocytes and an extracel-
lular matrix (ECM), which is largely made up of proteogly-
cans (PGs) and collagen. PGs are in turn composed by sul-
phated glycosaminoglycans (GAGs) linked to a protein core.
High levels of PGE2 mediate cartilage resorption by de-
creasing chondrocyte proliferation, inhibiting PG synthesis
and enhancing MMP activity. NO is one of the smallest bio-
logically active messenger molecules with a wide range of
biological actions. Increasing evidence indicates NO is in-
volved in the aetiopathogenesis of OA. The catabolic effect
of NO brings about the inhibition of PG synthesis and stimu-
lates chondrocytes to produce proenzymes which, following
conversion in their active form, cause cartilage breakdown
[6]. MMPs are a large family of calcium dependent, zinc-
containing endopeptidases, which are responsible for the
tissue remodelling and ECM degradation. Their expression
in arthritis diseases is modulated and upregulated by IL-1ꢀ
and TNF-ꢀ. In particular MMP-13, also known as colla-
genase-3, degrades native collagen fibrils (type I, II, and III)
and PG aggregates, showing the highest activity against type
II collagen, the main type of collagen in cartilage [7-9].
In physiological conditions, ECM undergoes a continu-
ous process of degradation and repair, whose balance is con-
trolled by chondrocytes, the only cell type present in articu-
lar cartilage, which are responsible for cartilage ECM com-
position.
In OA condition an imbalance between the degradation
and reparative/synthetic processes of the articular cartilage
occurs. This gives rise to changes of the physical properties
of the ECM attributable mainly to loss of collagen and PGs
as the result of the effects of different classes of catabolic
factors produced by chondrocytes, including proinflamma-
*Address correspondence to this author at the Dipartimento Farmaco-
chimico, Faculty of Pharmacy, University of Messina, Polo Universitario
dell’Annunziata, 98168 Messina, Italy; Tel: +39 090 6766408; Fax: + 39
090 6766402; E-mail: rottana@unime.it
1875-6638/13 $58.00+.00
© 2013 Bentham Science Publishers

5-Arylidene-4-Thiazolidinones Active as Antidegenerative Agents
Medicinal Chemistry, 2013, Vol. 9, No. 1 85
Ph
is implicated in the development of various inflammatory
pathologies. This enzyme catalyses the reduction of lipid
peroxidation-derived aldehydes, thus producing metabolites
which transduce inflammatory signalling by means of the
activation of protein kinases such as PKC and MAPK. These
in turn activate NF-kB that is responsible for the transcrip-
tion of many proinflammatory genes [4,5].
C3H7
O
Ph
N
(CH2)nOH
O
N
N
N
S
S
Ar
Ar
I
II
In the context of the complex inflammatory process in-
volved in the OA disease, it also appears to be useful to
evaluate the MMP-13 inhibitory activity of the compounds
under investigation.
Fig. (1). General structures of 5-arylidene-2-phenylimino-4-
thiazolidinones I, II.
1 Ar = 4-OC6H5-C6H4
2 Ar = 4-OH-C6H4
X
R
N
Currently intensive efforts are devoted to identify new
classes of MMP inhibitors with increased potency and selec-
tivity, including either molecules with more efficient zinc
binding groups or non-zinc binding compounds. Hydroxamic
acids act as potent chelating agents towards the zinc ion of
MMPs, but they can produce safety issues because of their
nonspecific action and toxicity. Although hydroxamates in-
teract with the MMP active site efficiently, the design of
suitable carboxylic acids as bioisosteres of hydroxamic acids
is considered an attractive strategy to discover new safer
MMP inhibitors. Many carboxylic acid-containing inhibitors
of MMPs, including MMP-13, were reported and some of
them are being evaluated in clinical trials [16-19]. The hy-
drophobicity has also been found to be an important deter-
minant in several classes of MMP inhibitors [17]. Among
various zinc-chelating agents active as MMP inhibitors, 2-
thioxo-4-thiazolidinones have been identified [18]. On these
bases, it is reasonable that carboxylic acid-containing 5-
arylidene-4-thiazolidinone derivatives 1-9 can act as biden-
tate zinc-chelators and thus they could be active as MMP
inhibitors.
S
3 Ar = 1-naphthyl
O
4 Ar = 4-OC6H5-C6H4
5 Ar = 4-OH-C6H4
Ar
1-9
6 Ar = 1-naphthyl
7 Ar = 3-OC6H5-C6H4
8 Ar = 4-OCH2C6H5-C6H4
9 Ar = 4-OH-C6H4
1-3 X = NPh; R = CH2C6H4COOH
4-6 X = O; R = CH2C6H4COOH
7-9 X = O; R = CH2COOH
Fig. (2). Structures of compounds 1-9.
We recently reported 5-arylidene-2-phenylimino-3-
propyl-4-thiazolidinones I (Fig. 1) which were found to be
endowed with interesting activity levels in models of acute
inflammation, such as carrageenan-induced paw and pleurisy
edema in rats, and to be inhibitors of COX isoforms [10].
Moreover, a series of N-hydroxyalkyl substituted 5-
arylidene-2-phenylimino-4-thiazolidinones II (Fig. 1) proved
to be able to counteract cartilage destruction during the in-
flammatory process by blocking the production or action of
catabolic factors induced by IL-1ꢀ[11].
MATERIALS AND METHODS
1. Chemistry
In light of the above, we here report the biological
evaluation of analogous 5-arylidene-2-phenylimino-4-thiazo-
lidinones (1-3, Fig. 2) and 5-arylidene-2,4-thiazolidinediones
(4-9, Fig. 2), which were already reported as potent inhibi-
tors of protein tyrosine phosphatase 1B (benzoic acids 1-6)
or aldose reductase (ALR2) (acetic acids 7-9) [12-15], in
order to assess their in vitro antidegenerative effect in culture
of human chondrocytes stimulated by IL-1ꢀ. Interestingly,
recent studies have demonstrated that ALR2 plays a pivotal
role in mediating oxidative stress-induced inflammation and
4-[(5-arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)me-
thyl]benzoic acids (1-3), 4-[(5-arylidene-2,4-dioxothia-
zolidin-3-yl)methyl]benzoic acids (4-6) and (5-arylidene-
2,4-dioxothiazolidin-3-yl)acetic acids (7-9) were synthesised
according to already reported procedures [12,13,15] which
are depicted in Schemes 1-3.
S
b
a
COOH
N
S
COOH
COOH
N
H
N
H
NCS
+
N
c
H2N
O
O
N
S
COOH
N
1
2
3
Ar = 4-OC6H5-C6H4
Ar = 4-OH-C6H4
Ar = 1-naphthyl
a) EtOH, ꢀ; b) ClCH2COCl, Et3N, EtOH, ꢀ; c) ArCHO, C5H11N, EtOH, ꢀ.
Ar
1-3
Scheme 1.

86 Medicinal Chemistry, 2013, Vol. 9, No. 1
Panico et al.
O
S
COOH
N
O
b
4 Ar = 4-OC6H5-C6H4
O
S
5
6
Ar = 4-OH-C6H4
Ar = 1-naphthyl
O
Ar
NH
a
NH
4-6
O
S
O
c, d
O
COOH
Ar
N
S
O
7
9
Ar = 3-OC6H5-C6H4
Ar = 4-OH-C6H4
Ar
7, 9
a) ArCHO, C5H11N, EtOH, ꢀ; b) BrCH2C6H4COOH, K2CO3, acetone, ꢀ;
c) BrCH2COOCH3, K2CO3, acetone, ꢀ; d) AcOH, HCl, ꢀ
Scheme 2.
O
S
O
S
COOH
COOH
O
O
S
N
N
NH
a, b
c
O
O
O
8
CH2Ph
a) BrCH2COOCH3, K2CO3, acetone, ꢀ; b) AcOH, HCl, ꢀ;
c) 4-PhCH2OC6H4CHO, C5H11N, EtOH, ꢀ.
Scheme 3.
4-[(5-Arylidene-4-oxo-2-phenyliminothiazolidin-3-yl)
and successive hydrolysis of methyl N-acetate intermediates
in concentrated hydrochloric acid and glacial acetic acid
(Scheme 2).
methyl]benzoic acids (1-3) were synthesized following the
synthetic pathway described in Scheme 1. The reaction of
phenylisothiocyanate and 4-(aminomethyl)benzoic acid in
ethanol at reflux provided the intermediate 4-(3-phenylthio-
ureidomethyl)benzoic acid which was converted into the 4-
[(4-oxo-2-phenyliminothiazolidin-3-yl)methyl]benzoic acid
by condensation with chloroacetyl chloride in the presence
of triethylamine as base. According to the Knoevenagel pro-
cedure, the condensation of the 4-oxo-2-phenylimino-
thiazolidine derivate with the appropriate aromatic aldehydes
provided compounds 1-3, using piperidine as base.
Because of the susceptibility to hydrolysis of the 4-
benzyloxy group, the synthesis of compound 8 required a
different synthetic procedure. In fact, the first synthetic step
involved the N-alkylation of the commercially available 2,4-
thiazolidinedione with methyl bromoacetate followed by
acidic-catalyzed hydrolysis of the methyl acetate portion
afforded (2,4-dioxothiazolidin-3-yl)acetic acid. The latter
compound was converted to compound 8 by condensation
with 4-benzyloxy benzaldehyde.
Scheme 2 reports the general synthesis of 4-thiazo-
lidinediones 4-7 and 9. The appropriate 5-aryliden-2,4-tiazo-
lidinedione intermediates were obtained by condensation of
the commercially available 2,4-tiazolidinedione and properly
substituted aldehydes, in ethanol at reflux in the presence of
piperidine as base. The subsequent N-alkylation of the ap-
propriate intermediates with 4-bromomethylbenzoic acid
under basic conditions provided compounds 4-6 with good
yields.
In all cases the introduction of the 5-arylidene moiety on
4-thiazolidinone scaffold provided only Z isomers. The
structures of tested compounds were characterized by means
of analytical, 1H and 13C NMR data.
2. MMP-13 Fluorimetric Assay
The compounds were evaluated for their ability to inhibit
the hydrolysis of fluorescence-quenched peptide substrate
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Biomol, Inc.).
The assays were performed in 50 mM HEPES buffer con-
taining 5 mM CaCl2, 0.1mM ZnCl2, 0.05% Brij-35, at pH 7,
The corresponding acetic acids 7 and 9 were yielded by
N-alkylation of appropriate 5-arylidene-2,4-thiazolidi-
nediones with methyl bromoacetate under basic conditions

5-Arylidene-4-Thiazolidinones Active as Antidegenerative Agents
Medicinal Chemistry, 2013, Vol. 9, No. 1 87
using 10 nM of proteolytic enzyme (catalytic domains of
MMP-13) (Biomol, Inc.) and 350 nM of peptide. The en-
zyme was incubated at 25°C with increasing concentration
densities of standard solutions of sodium nitrite standard
curve (0–120 ꢀM).
of the inhibitor and the fluorescence (excitation
328 nm;
6. Determination of Glycosaminoglycans (GAGs)
max
emission max 393 nm) was measured for 3 min after the addi-
tion of the substrate using a Varian Eclipse fluorimeter. Fit-
ting of rates as a function of inhibitor concentration provided
IC50 values. The inhibitor N-isobutyl-N-[4-methoxyphenyl-
sulfonyl]glycyl hydroxamic acid (Biomol, Inc.) was used as
control [19].
The level of GAGs, an index of cartilage damage, was
measured by spectrophotometry with a solution of 1,9-
dimethylmethylene blue at ꢁ=535 nm using the method of
Farndale et al. [22]. The amount of glycosaminoglycans was
calculated from a standard curve (100–500 ꢀg/ml) obtained
for shark chondroitin sulphate C, derived from shark carti-
lage.
3. Cell Viability Assay
The cytotoxic effect of the tested substances was evalu-
ated by a cell viability test based on the cleavage of 3-(4,5-
7. Statistical Analysis
All the present results are means S.E.M. of three experi-
ments performed on quadruplicate samples. The Student’s t-
test was used to evaluate the differences between the means
of each group. P < 0.05 was considered to be statistically
significant. The statistical analysis was performed by using
one-way ANOVA followed by Dunnett’s post-hoc test for
multiple comparison with control. All statistical analyses
were performed using the statistical software package SYS-
TAT, version 9 (Systat Inc., Evanston IL, USA).
dimethylthiazol-2-yl)-2.5-diphenyltetrazolium
bromide
(MTT) by mitochondrial dehydrogenases of metabolically
active cells [20].
4. Human Articular Chondrocyte Culture
Normal human articular cartilage was obtained at re-
placement surgery from some patients with femoral neck
accidental fractures and that informed consent was obtained.
The isolation procedure was conducted under antiseptic
conditions. The cartilage was cut into small fragments and
carefully washed using Dulbecco’s Modified Eagles Me-
dium (DMEM) culture medium containing NaHCO3, 25 mM
Hepes, 1 mM sodium pyruvate, 50 mg/ml gentamycin, 100
U/ml penicillin, 100 mg/ml streptomycin and 2.5 mg/ml am-
photericin B. Chondrocytes were isolated through three se-
quential passages of enzymatic digestion of the extracellular
matrix: incubation with 0.1% hyaluronidase type III (1
mg/ml for 100 mg of cartilage), for 30 min at 37 °C; incuba-
tion with 0.5% pronase type XIV (5 mg/ml for 100 mg of
cartilage), for 60 min at 37 °C; finally incubation with 0.2%
collagenase type IA (2 mg/ml for 100 mg of cartilage), for
45 min at 37 ° C. The obtained cellular suspension was fil-
tered (filters from 100 and 70 mm) to eliminate the residues
of the digestion, cellular aggregates. Freshly isolated chon-
drocytes were seeded into monolayer culture at a cell density
of 2 ꢁ 105 cells and cultured in 1 ml of DMEM supple-
mented with 10% foetal bovine serum (FCS) at 37°C in 5%
CO2/95% air. Confluent chondrocytes of primary culture
were used for all the experiments: the tested compounds
were assayed dissolved in DMSO, appropriately diluted in
Dulbecco’s Modified Eagle’s Medium (DMEM) and dis-
pensed to the wells treated as follows: Control, IL-1ꢀ (10
ng/ml), indomethacin (10, 2.5 and 0.01 ꢀg/ml) + IL-1ꢀ (10
ng/ml), 1–9 (10, 2.5 and 0.01ꢀg/ml) + IL-1ꢀ (10 ng/ml).
After 120 h the supernatants of cartilage culture were col-
lected for different assays.
RESULTS AND DISCUSSION
Table 1 reports the results of the MMP-13 inhibitory ac-
tivity of thiazolidinones 1-9. Out of the tested compounds, 4-
{[(5-naphthalen-1-ylmethylidene)-2,4-dioxothiazolidin-3-
yl]methyl}benzoic acid (6) proved to be the most potent
MMP-13 inhibitor (IC50 = 0.11 ꢀM), followed by 2-
phenylimino substituted analogue 1 (IC50 = 1.7 ꢂM) and 2,4-
thiazolidinedione 5 (IC50 = 4.8 ꢀM). Both 4-[(5-arylidene-
2,4-dioxothiazolidin-3-yl)methyl]benzoic acids 5 and 6, 4-
hydroxyphenyl- and 1-naphthyl substituted respectively,
exhibited significantly higher potency than their 2-
phenylimino counterparts 2 and 3, whereas out of the 4-
phenoxybenzylidene substituted analogues (1 and 4) 2-
phenylimino derivative 1 was almost 70-fold more active
than the corresponding 2,4-thiazolidinedione 4.
Therefore, in contrast with other classes of MMP inhibi-
tors, the lipophilicity of these compounds did not appear to
be
a
determinant feature, since 5-naphthalen-1-
ylmethylidene-2-phenylimino-4-thiazolidinone 3, which is
more lipophilic than 6, did not prove to be efficacious (Table
1). On the contrary, 2-phenylimino derivative 1 was re-
markably more active than its 2,4-thiazolidinedione counter-
part 4. Among 5-(4-hydroxybenzylidene) substituted deriva-
tives (2 and 5), 2,4-thiazolidinedione 5 was the most effica-
cious. The removal of the phenyl ring in the acidic moiety on
N-3 of benzoic acid 5 led to a 10-fold less potent inhibitor
(acetic acid 9), which, however, showed activity similar to
that of analogous 4-hydroxybenzylidene substituted benzoic
acid 2. Comparing the inhibitory effects of 4-hydroxyphenyl
substituted analogues 5 and 9 suggested that the p-methyl
benzoic acid residue in position 3 might be more favourable
than the acetic chain for MMP-13 inhibition.
5. Determination of Nitrite
Nitrite was determined by adding 100 ꢀl of Griess rea-
gent (1% sulphanylamide and 0.1% naphthylethylenedia-
mine dihydrochloride in 5% of hydrochloric acid) to 100 ꢀl
of samples [21]. The optical density at ꢀ = 570 nm was
measured using a microtiter plate reader. Nitrite concentra-
tions were calculated by comparison with respective optical
The antidegenerative effect of compounds 1-9 was
evaluated at different concentrations (10, 2.5 and 0.01
ꢀg/ml) in culture of human chondrocytes stimulated by IL-
1ꢀ. The results are reported at 10 ꢀg/ml and 2.5 ꢀg/ml, since

88 Medicinal Chemistry, 2013, Vol. 9, No. 1
Panico et al.
Table 1. In vitro MMP-13 Inhibitory Activity and Calculated LogP Values of Compounds 1-9
Compd
R
X
Ar
IC50 (ꢀM)
LogPa
1
CH2C6H4COOH
CH2C6H4COOH
CH2C6H4COOH
CH2C6H4COOH
CH2C6H4COOH
CH2C6H4COOH
CH2COOH
NC6H5
4-OC6H5-C6H4
4-OH-C6H4
1.7
53.22
n.a.
7.02
5.21
6.51
4.96
3.15
4.45
3.05
3.12
1.25
2
NC6H5
3
NC6H5
1-naphthyl
4
O
O
O
O
O
O
4-OC6H5-C6H4
4-OH-C6H4
109
5
4.8
6
1-naphthyl
0.11
11.1
22.3
48
7
3-OC6H5-C6H4
4-OCH2C6H5-C6H4
4-OH-C6H4
8
9
CH2COOH
CH2COOH
NNGH
0.0026
a Marvin Calculator Plugins, http://www.chemaxon.com.
n.a. = no activity at 100 ꢀM.
NNGH = N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid.
Representative results of at least 3 independent experiments are reported.
a NO production (ꢀM) (means SEM) in the culture medium by IL-1ꢁ stimulated articular chondrocytes 120 h after the addition of compounds 1-9 at 2.5
and 10 ꢀg/ml.
* Significantly different from IL-1ꢁ treated samples (P < 0.005).
Indo = indomethacin.
Fig. (3). Effect of 5-arylidene-4-thiazolidinone derivatives 1-9 on NO productiona.
the activity proved to be negligible at 0.01 ꢀg/ml. Indo-
methacin was used as reference drug.
they did not interfere with cell viability at tested doses (data
not shown).
The tetrazolium dye (MTT) fluorogenic assay was cho-
sen as the method to assess cell viability. MTT assay showed
that all tested compounds did not reduce the ability of chon-
drocytes to metabolise tetrazolium salts, demonstrating that
Fig. (3) reports the NO release obtained 120 h after the
addition of compounds 1-9, at the concentrations of 2.5
ꢃg/ml and 10 ꢃg/ml. It can be observed that NO production
in chondrocytes under basal condition is low, mainly due to

5-Arylidene-4-Thiazolidinones Active as Antidegenerative Agents
Medicinal Chemistry, 2013, Vol. 9, No. 1 89
a GAG release (ꢀg/ml) (means SEM) in the culture medium by IL-1ꢁ stimulated articular chondrocytes 120 h after the addition of compounds 1-9 at 2.5 and
10 ꢀg/ml.
* Significantly different from IL-1ꢁ treated samples (P < 0.005).
Indo = indomethacin.
Fig. (4). Effect of 5-arylidene-4-thiazolidinone derivatives 1-9 on GAG releasea.
the activity of the constitutive NO synthase. When human
chondrocytes were treated with IL-1ꢀ, a substantial increase
in the NO production was observed. The tested compounds
as well as indomethacin, when combined with IL-1ꢀ, pro-
vided a significant reduction in the NO release compared to
the samples treated only with IL-1ꢀ. All compounds 1-9
were able to decrease the NO production at both 2.5 ꢀg/ml
and 10 ꢀg/ml concentrations, except compounds 3 and 5 that
produced a significant reduction in NO release only at the
highest concentration (10 ꢀg/ml). Interestingly, benzoic acid
4 and acetic acids 7-9 proved to be the most efficacious de-
rivatives restoring NO levels similar to those of control sam-
ples at both selected concentrations. Moreover, all com-
pounds 1-9 proved to be more effective than indomethacin at
10 ꢀg/ml dose and, among them, compounds 4, 7-9 were
more active than the reference drug also at 2.5 ꢀg/ml dose.
CONCLUSIONS
In conclusion, the biological evaluation here reported in-
dicated that 5-arylidene-4-thiazolidinone derivatives 1-9 can
stimulate the GAG production and reduce NO release by
chondrocytes, similarly to previously investigated analogues
[11], thus suggesting that the tested compounds could block
multiple cartilage destruction during the arthritic inflamma-
tory process. In particular, acetic acids 7-9 showed apprecia-
ble chondroprotective activity, since they were the most effi-
cacious derivatives in normalizing NO levels, despite they
resulted to be only moderate MMP-13 inhibitors. Benzoic
acids 1, 5 and 6 proved to be effective MMP-13 inhibitors at
low micromolar doses and they were also able to restore
normal levels of GAGs showing to exert interesting antide-
generative effects.
In the context of these results, it appears of interest that
compounds 7-9 are also potent ALR2 inhibitors. Our results
suggest that the activity of these compounds might be corre-
lated to their ALR2 inhibitory ability by preventing the
stimulation of PKC or MAPK and the subsequent activation
of NF-kB. On the whole, the promising antidegenerative
profile of these 5-arylidene-4-thiazolidinone derivatives en-
courages the evaluation of other analogues as well as the
study of their action mechanism and activity in other cell
types involved in inflammatory lesions.
On the whole, the (2,4-dioxothiazolidin-3-yl)acetic acid
moiety of compounds 7-9 appeared to be favourable in order
to decrease the NO levels in inflammatory conditions,
whereas the 5-arylidene group resulted to be scarcely rele-
vant.
Moreover, the treatment of chondrocytes with IL-1ꢀ
proved to significantly reduce GAG production (Fig. 4). At
10 ꢀg/ml, all compounds 1-9 protected IL-1ꢀ-treated chon-
drocytes, which were able to restore normal levels of GAGs
available for the synthesis of PGs. Moreover, analogously to
indomethacin, the treatment with compounds 8 and 9 proved
to achieve GAG basal levels also at 2.5 ꢀg/ml dose.ꢀꢀ
CONFLICTS OF INTEREST
The author(s) confirm that this article content has no con-
flicts of interest.
Overall, among the tested compounds, acetic acid deriva-
tives 7-9 showed the most promising activity, since they
were able both to reduce NO amount to basal levels and to
restore normal GAG production, also at the lowest tested
concentration. Interestingly, their potency in reducing NO
production resulted to be higher than that of indomethacin.
ACKNOWLEDGEMENT
Declared none.

90 Medicinal Chemistry, 2013, Vol. 9, No. 1
Panico et al.
[9]
Forsyth, C.B.; Cole, A.; Murphy, G.; Bienias, J.L.; Loeser, R.F.
Increased matrix metalloproteinase-13 production with aging by
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ABBREVIATIONS
COX
ECM
GAGs
IL-1ꢀ
MMPs
NF-kB
NO
=
=
=
=
=
=
=
=
=
=
=
=
cyclooxygenase
[10]
Ottanà, R.; Maccari, R.; Barreca, M.L.; Bruno, G.; Rotondo, A.;
Rossi, A.; Chiricosta, G.; Di Paola, R.; Sautebin, L.; Cuzzocrea, S.;
Vigorita, M.G. 5-Arylidene-2-imino-4-thiazolidinones: design and
synthesis of novel anti-inflammatory agents. Bioorg. Med. Chem.,
2005, 13, 4243-4252.
extracellular matrix
glycosaminoglycans
interleukin-1ꢀ
[11]
Ottanà, R.; Maccari, R.; Ciurleo, C.; Vigorita, M.G.; Panico, A.M.;
Cardile, V.; Garufi, F.; Ronsisvalle, S. Synthesis and in vitro eva-
matrix metalloproteinases
nuclear factor kB
nitric oxide
luation
of
5-arylidene-3-hydroxyalkyl-2-phenylimino-4-
thiazolidinones with antidegenerative activity on human chondro-
cyte cultures. Bioorg. Med. Chem., 2007, 15, 7618-7625.
Maccari, R.; Paoli, P.; Ottanà, R.; Jacomelli, M.; Ciurleo, R.; Ma-
nao, G.; Steindl, T.; Langer, T.; Vigorita, M.G.; Camici, G. 5-
Arylidene-2,4-thiazolidinediones as inhibitors of protein tyrosine
phosphatases. Bioorg. Med. Chem., 2007, 15, 5137-5149.
Ottanà, R.; Maccari, R.; Ciurleo, R.; Paoli, P.; Jacomelli, M.; Ma-
nao, G.; Camici, G.; Laggner, C.; Langer, T. 5-Arylidene-2-
phenylimino-4-thiazolidinones as PTP1B and LMW-PTP inhibi-
tors. Bioorg. Med. Chem., 2009, 17, 1928-1937.
[12]
[13]
[14]
[15]
[16]
OA
osteoarthritis
PGE2
PGs
prostaglandins E2
proteoglycans
ROS
reactive oxygen species
tumor necrosis factor- ꢀ
Bruno, G.; Costantino, L.; Curinga, C.; Maccari, R.; Monforte, F.;
Nicolò, F.; Ottanà, R.; Vigorita, M.G. Synthesis and aldose reduc-
tase inhibitory activity of 5-arylidene-2,4-thiazolidinediones. Bio-
org. Med. Chem., 2002, 10, 1077-1084.
TNF-ꢀ
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Received: May 15, 2011
Revised: April 29, 2012
Accepted: May 28, 2012