Synthesis and in vitro evaluation of 5-arylidene-3-hydroxyalkyl-2-phenylimino-4-thiazolidinones with antidegenerative activity on human chondrocyte cultures

Article abstract of DOI:10.1016/j.bmc.2007.09.001

5-Arylidene-3-hydroxyalkyl-2-phenylimino-4-thiazolidinones (7,8) were synthesized and evaluated for their antidegenerative activity on human chondrocyte cultures stimulated by IL-1β. This in vitro model has proven to be a useful experimental model to repr

Full text of DOI:10.1016/j.bmc.2007.09.001

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7618–7625
Synthesis and in vitro evaluation
of 5-arylidene-3-hydroxyalkyl-2-phenylimino-4-thiazolidinones
with antidegenerative activity on human chondrocyte cultures
a,
a
a
a
*
`
Rosaria Ottana, Rosanna Maccari, Rosella Ciurleo, Maria Gabriella Vigorita,
Anna Maria Panico,b Venera Cardile,c Floriana Garufib and Simone Ronsisvalleb
a
`
Dipartimento Farmaco-chimico, Universita di Messina, Polo Universitario dell’Annunziata, 98168 Messina, Italy
b
`
Dipartimento di Scienze Farmaceutiche, Universita di Catania, V.le A. Doria 6, 95125 Catania, Italy
Dipartimento di Scienze Fisiologiche, Universita di Catania, V.le A. Doria 6, 95125 Catania, Italy
c
`
Received 15 June 2007; revised 30 August 2007; accepted 4 September 2007
Available online 8 September 2007
Abstract—5-Arylidene-3-hydroxyalkyl-2-phenylimino-4-thiazolidinones (7,8) were synthesized and evaluated for their antidegener-
ative activity on human chondrocyte cultures stimulated by IL-1b. This in vitro model has proven to be a useful experimental model
to reproduce the mechanisms involved in arthritic diseases. The cell viability, the amount of GAGs, the production of NO and PGE2
and the inhibition of MMP-3 were measured. Several thiazolidinones 7 and 8 exhibited the ability to block the production or action
of the degenerative factors induced by IL-1b.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
which IL-1b proves to be significant. It induces high lev-
els of proinflammatory mediators, such as prostaglan-
dins E2 (PGE2) and nitric oxide (NO), and inhibits
collagen and PG synthesis. IL-1b is a pivotal driving
force in inducing and sustaining cartilage damage that
usually leads to the loss of sulfated glycosaminoglycans
(GAGs).2 GAG release is a consequence of increased
matrix protease activity, leading to the cleavage of colla-
gen and PGs. Thus, GAGs can be considered an indica-
tor of catabolic events in cartilage.
Osteoarthritis (OA) is one of the most common chronic
diseases and its management represents an enormous
cost to the health care system. OA is a multifunctional
disease in which the principal target of the pathogenic
process is the articular cartilage. Cartilage consists of a
relatively small number of chondrocytes and many extra-
cellular matrix (ECM) components, which comprise
mainly collagen fibrils and aggrecan (a large aggregating
proteoglycan). Chondrocytes synthesize and catabolize
ECM macromolecules, while the matrix in turn functions
to maintain the homeostasis of the cellular environment
and the cartilage structure. In diseases such as OA the
degradation of the ECM exceeds its synthesis, thereby
resulting in a net decrease in the amount of cartilage ma-
trix that depletes aggrecan. In addition, the disregulated
synthesis of matrix components, such as proteoglycans
(PGs) and collagen, plays a fundamental role.1
High levels of PGE2 also mediate cartilage resorption by
decreasing chondrocyte proliferation, enhancing matrix
metalloproteinase (MMP) activity and inhibiting aggre-
can synthesis in chondrocytes.3 Several classes of MMPs
have been implicated in tissue destruction: MMP-1
(interstitial collagenase), MMP-3 (stomelysin-1) and
MMP-9 (gelatinase-B). These enzymes produced by
chondrocytes have been reported to play a significant
role in the destruction of the cartilage matrix in arthritic
diseases. MMP-3 is particularly able to degrade matrix
components including the core protein of the PGs.4
Moreover, the functional alteration of cartilage also re-
sults from the intervention of different cytokines, among
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
widely used in the long term treatment of inflammatory
diseases, mainly for arthritic pain. They inhibit
cyclooxygenase (COXs, also known as prostaglandin
Keywords: 2-Phenylimino-4-thiazolidinones; Osteoarthritis; Human
chondrocyte; Antidegenerative activity.
*
Corresponding author. Tel.: +39 90 6766408; fax: +39 90 355613;
e-mail: ottana@pharma.unime.it
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.001

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7619
synthetase), which is the key enzyme in prostaglandin
biosynthesis from arachidonic acid. Two isoforms of
COX have been characterized: COX-1, a constitutive
form that plays a cytoprotective role in the gastro-intes-
tinal tract, and COX-2, a form generally induced in
inflammatory sites.5
In light of the above we here report the biological
evaluation of a class of newly synthesized 5-arylidene-
2-phenylimino-4-thiazolidinones, 3-hydroxyethyl (7)
and 3-hydroxypropyl (8) substituted, in order to evalu-
ate how is their in vitro anti-inflammatory and protec-
tive effect on inflammatory degenerative diseases as
OA compared with 2. A hydroxyl group on different al-
kyl chains in position 3 of the 4-thiazolidinone moiety
was introduced in order to both increase their hydrophi-
licity and establish additional bonds with the biological
target. The substituents on the 5-arylidene moiety were
selected on the basis of the SARs which we previously
acquired by means of in vivo anti-inflammatory assays.9
Recently, it has been suggested that a number of NSA-
IDs might accelerate joint damage, probably as a result
of inhibitory effects on cartilage PG synthesis or by
enhancing production of cartilage bone-destructive
IL-1b. In addition, several reports have demonstrated
that some NSAIDs may also modify cytokine
production.6
We decided to investigate particularly the chondropro-
tective effect of selected compounds 7 and 8 in cultures
of human chondrocytes stimulated by IL-1b in an
in vitro model that has proven to be a useful experimen-
tal model to reproduce the mechanisms involved in ar-
thritic diseases.10 The cell viability, the amount of
GAGs, the production of NO and PGE2 and the inhibi-
tion of MMP-3 were measured at different concentra-
tions (10, 50 and 100 lg/mL). Indomethacin was used
as reference drug.
Nowadays the main goal of research in this field is to
find new anti-inflammatory drugs that are safer for long
term use and that have a chondroprotective effect.
For a long time we have studied 4-thiazolidinone
derivatives because of their interesting in vivo anti-
inflammatory/analgesic properties along with a better
gastrointestinal safety profile than those of known NSA-
IDs.7 Moreover, some derivatives have been shown to
be preferential inhibitors of COX-2 by suitable selectiv-
ity assays and supported by molecular modelling
studies.8
2. Chemistry
As a part of an ongoing programme to design safer
NSAIDs, 5-arylidene-2-phenylimino-4-thiazolidinones
(1) proved to be interesting in vivo antiinflammatory
compounds that reduce the carrageenan-induced paw
and pleurisy oedema in rats. In the carrageenan-induced
paw oedema assay, 5-(3-methoxybenzylidene)-2-phe-
nylimino-3-propyl-4-thiazolidinone (2) was found to
reach an inhibition level similar to that of indomethacin
and had better activity profile than that of celecoxib
(Fig. 1).9
Compounds 7–8 (a–e) were synthesized according to the
synthetic pathway described in Scheme 1. The synthesis
of 2-phenylimino-4-thiazolidinones 511 and 6 was
performed by condensation of N-hydroxyethyl- or
N-hydroxypropyl-N0-phenylthiourea (3, 4)12,13 with
methyl bromoacetate in the presence of triethylamine
in ethanol at reflux for 24 h. The reaction provided only
3-hydroxyalkyl isomers 5 or 6; however, when the reac-
tion was carried out at room temperature in CHCl3,
both the phenylimino and hydroxyalkylimino isomers
Ph
R
N
S
S
NH2SO2
i
CH2COOH
N
MeO
BrCH2COOCH3
Ph
N
H
N R
H
+
O
N
N
CH3
CF3
N
3, 4
5, 6
O
H3C
ii
Cl
Ph
Celecoxib
Indomethacin
R
N
S
3, 5, 7: R = (CH2)2OH
4, 6, 8: R = (CH2)3OH
N
O
N
R'
N
CH2CH2CH3
O
R
R' : a 4-SCH3
b 4-SO2CH3
N
N
S
S
c 3-OCH3
d 4-Cl
O
7, 8 (a-e)
Ar
e 4-OCH3
1
2
MeO
Scheme 1. Reagents: (i) Et3N, EtOH, D 24 h; (ii) ArCHO, piperidine,
EtOH, D 24 h.
Figure 1.

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7620
were isolated. In fact two possible reaction products
could originate from the condensation of methyl bro-
moacetate with the sulfur atom of two different interme-
diate thiols generated from thioureas (3, 4) by
delocalization of the lone pairs of the two different nitro-
gens on the adjacent thiocarbonyl group. The type of
solvent, the temperature and the thioureas substituents
proved to be determinant for the reaction.9
and 100 lg/mL. It can be observed that when the tested
compounds were combined with IL-1b this provided a
significant reduction in NO release compared to the
samples treated with IL-1b. In particular, compounds
7d, 8d and 8e as well as compounds 7b and 8a at all
tested concentrations proved to be the most efficacious
derivatives as levels of NO production were lower than
control samples (4.61 lM) and also indomethacin
(7.56 lM). Such a result might be related to the inhibi-
tion of constitutive NO synthetase, an enzyme consid-
ered to be the mainly responsible for NO production
under basal conditions.
5-Arylidene-2-phenylimino-3-hydroxyalkyl-4-thiazolidi-
nones 7 and 8 were synthesized by the reaction in basic
conditions of compounds 5 or 6 and appropriate alde-
hydes in refluxing ethanol for about 24 h. Compounds
7b and 8b were synthesized by oxidation with hydrogen
peroxide in acetic acid of corresponding methylthio
derivatives 7a and 8a, respectively.
3.3. Determination of GAGs
The total concentration of GAGs, an index of cartilage
damage, in the medium of chondrocyte culture, is pre-
sented in Figure 4. It can be seen that the tested com-
pounds combined with IL-1b showed a significant
inhibition of IL-1b activity. At 100 lg/mL all the tested
compounds by preventing the depletion of proteoglycan
reported values similar to those of the untreated controls.
The structure of all compounds was assessed by elemen-
1
tal analysis, H and 13C NMR. The resonances of alkyl
protons allowed us to identify the isomer structure and
by analogy of previously reported compounds. The
NCH2 protons appeared as a triplet resonating at chem-
ical shift ranging between 3.93 ppm (5) and 3.65 ppm
(6), due to the deshielding effect generated by the
extended electronic delocalization of 3-N lone pair. In
fact, the same protons of the alkylimino isomers reso-
nated at lower chemical shifts.9
3.4. Determination of MMP-3
The results of the MMP-3 production are given in Fig-
ure 5. This study made it possible to demonstrate the
ability of the tested compounds to suppress IL-1b-in-
duced MMP-3 secretion in human articular chondro-
cytes. At 50 lg/mL all tested compounds significantly
reduced MMP-3 secretion level reaching low values even
compared with indomethacin. In particular, compounds
7a, 7d and 8b achieved the greatest decrease of the
MMP-3 secretion.
In 1H NMR spectra of 5-arylidene-4-thiazolidinones
derivatives (7, 8), the NCH2 protons appeared as a
triplet resonating at chemical shift ranging between
4.01–3.90 ppm (7) and 3.68–3.66 ppm (8). Moreover,
the absence of the signal of 5-CH2 protons of starting
compounds (5, 6) together with the resonance of the
methine hydrogen (7.72–7.93 ppm) agreed with the
structures proposed above. Moreover, 13C spectra
showed a clear change in the splitting pattern of 5-C
which resonated as a triplet in 5 and 6 and as a singlet
in compounds 7 and 8.
3.5. Determination of PGE2
The cells treated with IL-1b (10 ng/mL) released signifi-
cant levels of PGE2. All tested compounds 2, 7 and 8
reduced PGE2 production without reaching the inhibi-
tion level of indomethacin (40.07 ng/mL). Compound
8b was found to be the most active compound
(53.22 ng/mL at 100 lg/mL) (Fig. 6).
1H NMR spectra and X-ray crystallography of a previ-
ously reported analogue allowed us to assign the Z con-
figuration of the exocyclic C@C bond. In particular the
methine proton resonance (7.73–7.77 ppm) became
diagnostic; in fact it resonated at lower chemical shifts
than E isomers owing to a greater deshielding effect of
the adjacent C@O than that of 1-S.14
4. Discussion
OA appears to be the result of an imbalance between the
destructive and reparative/synthetic processes of the
articular cartilage, in which some substances as free
radicals (ROS and NO), inflammatory cytokines (e.g.,
IL-1 and TNF-a) and metalloproteinases (MMP) are
produced in excess.15,16 Therefore inhibitors of these
mediators could represent a new type of chondroprotec-
tive drug for treating OA diseases. The above results
showed that the protective activity of the tested com-
pounds (2, 7 and 8), assayed at different concentrations
(10, 50 and 100 lg/mL), generally was dose dependent.
3. Results
3.1. Cell viability assay
MTT assay showed that all tested compounds did not
reduce the ability of chondrocytes to metabolise tetrazo-
lium salts, demonstrating that they did not interfere with
cell viability at all tested concentrations (Fig. 2).
3.2. Determination of nitrite
They strongly inhibited NO production induced by
inflammatory IL-1b (Fig. 3). Both compounds 7 and 8
showed the same effect in reducing the IL-1b induced
NO production independently of groups on 3-N, while
Figure 3 shows NO production levels by IL-1b stimu-
lated articular chondrocytes 120 h after the addition of
compounds 2, 7 and 8, at the concentrations of 10, 50

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7621
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control
Indom+IL
7a+IL
7b+IL
7c+IL
7d+IL
7e+IL
8a+IL
8b+IL
8c+IL
8d+IL
8e+IL
2+IL
Figure 2. Cell viability (means SEM) in the culture medium by articular chondrocytes 120 h after the addition of compounds 2, 7–8 (a–e) at 10, 50
and 100 lg/mL, respectively. Values are expressed as Abs (absorbance).
14
12
10
8
6
4
2
0
Control
IL
Indom+IL
7a +IL
7b +IL
7c +IL
7d +IL
7e +IL
8a +IL
8b +IL
8c +IL
8d +IL
8e +IL
2 +IL
Figure 3. NO production (lM) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes 120 h after the addition of
compounds 2, 7–8 a–e at 10, 50 and 100 lg/mL, respectively. *Significantly different from IL-1b treated samples (P < 0.005).
400
350
300
250
200
150
100
50
0
Control
IL
Indom+IL
7a +IL
7b +IL
7c +IL
7d +IL
7e+ IL
8a +IL
8b +IL
8c +IL
8d +IL
8e +IL
2 +IL
Figure 4. GAGs release (lg/mL) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes 120 h after the addition of
compounds 2, 7–8 a–e at 10, 50 and 100 lg/mL, respectively. *Significantly different from IL-1b treated samples (P < 0.005).
the substituents on 5-arylidene moiety appeared to influ-
ence the activity. Of the tested compound 8d was the
most active followed by 7d showing that the chlorine
atom was beneficial for activity; its replacement with
OCH3 was indifferent in the case of 8e while it showed
to be detrimental in the case of 7e. The replacement of
OCH3 with SCH3 resulted to be beneficial for com-
pound 7a but 8a was less active at all tested concentra-
tions. The oxidation of 7a and 8a to the corresponding
sulfones (7b, 8b) produced the same effect. The insertion
of the OCH3 group to the meta position (7c, 8c and 2)
increased NO production levels thus having a detrimen-
tal effect on activity.
In the present study we observed that the tested 5-arylid-
ene-2-phenylimino-4-thiazolidinones reduced the IL-1b-
induced PG depletion as well as MMP-3 production
(Figs. 4 and 5). In particular compounds 7 and 8
decreased the production of MMP-3, which ranged
between 43.76 and 88.77 ng/mL at 100 and 50 lg/mL,

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7622
200
180
160
140
120
100
80
60
40
20
0
Control
IL
Indom+IL
7a +IL
7b +IL
7c +IL
7d +IL
7e +IL
8a +IL
8b +IL
8c +IL
8d +IL
8e +IL
2 +IL
Figure 5. MMP-3 production (ng/mL) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes 120 h after the addition of
compounds 2, 7–8 a–e at 10, 50 and 100 lg/mL, respectively. *Significantly different from IL-1b treated samples (P < 0.005).
100
90
80
70
60
50
40
30
20
10
0
Control
IL
Indom +IL
7a +IL
7b +IL
7c +IL
7d + IL
7e +IL
8a +IL
8b +IL
8c +IL
8d +IL
8e +IL
2 +IL
Figure 6. PGE2 production (ng/mL) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes 120 h after the addition of
compounds 2, 7–8 a–e at 10, 50 and 100 lg/mL, respectively. *Significantly different from IL-1b treated samples (P < 0.005).
respectively, in comparison with indomethacin (62.64
ng/mL). In particular at 100 lg/mL 7b, 7c, 8a, 8c, 8d,
8e and 2 exhibited levels of MMP-3 production similar
to that of indomethacin, whereas 7a, 7d and 8b proved
to be even more active. The ability to reduce MMP-3 en-
zyme appeared to be linked to the 5-arylidene-2-phenyli-
mino-4-thiazolidinone skeleton, but to be independent
of both N-3 and 5-arylidene substituents.
5-arylidene-2-phenylimino-4-thiazolidinones (2, 7–8) are
attributable to their ability to block the production or
action of the degenerative factors induced by IL-1b.
This may be beneficial in the treatment of arthritic
disease.
Comparing the activity of compound 2 with 7 and 8
analogues indicates that the introduction of 3-hydrox-
yalkyl groups produces only minor effects on the antide-
generative activity of human chondrocytes. They
generally show similar ability to block cartilage destruc-
tion except for the release of NO in the culture medium.
In fact, compounds 7 and 8 significantly reduce NO
production in comparison with 2.
As regards the effect on GAGs, the treatment with all
compounds reduced the catabolic response induced by
proinflammatory cytokine. The best chondroprotective
compound was 7a (339.25 lg/mL at 100 lg/mL).
It is known that IL-1b mediates the inflammatory re-
sponse via the stimulation of PGE2 production. In our
experiments, it can be observed that PGE2 production
was inhibited by all the tested thiazolidinones, without
reaching the activity levels of indomethacin in any case.
The most active compound was 8b.
On the whole the antidegenerative activity of the
selected thiazolidinones shows to be attributable to 5-
arylidene-2-phenylimino-4-thiazolidinone system that
appears promising for the development of novel attrac-
tive anti-inflammatory/antidegenerative agents by
blocking cartilage destruction during the inflammatory
process.
5. Conclusions
Further insights into the structural and biological prop-
erties of the compounds are in the process of being
examined.
Overall, the experimental results here reported led us
to suggest that the anti-inflammatory properties of

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7623
6. Experimental
J = 4.8 Hz); 6.92–7.44 (m, 9H, arom); 7.76 (s, 1H,
CH); 13C NMR (CDCl3):
46.1 (NCH2); 55.2
d
6.1. Chemistry
(OCH3); 61.6 (CH2OH); 115.3, 115.5, 121.3, 122.1,
125.2, 129.4, 131.7 (CH arom); 121.6 (5-C); 130.4 (CH
methylidene); 134.8, 147.3, 152,5, 159,9 (Cq); 167.1
(CO); Anal. (C19H18N2O3S) C, H, N.
Melting points were recorded on a Kofler hot-stage appa-
ratus and are uncorrected. TLC controls were carried out
on precoated silica gel plates (F 254 Merck). Elemental
analyses (C, H, N), determined by means of a C. Erba
mod. 1106 elem. Analyzer, were within 0,4% of theory.
1H and 13C NMR spectra were recorded on a Varian 300
6.3.3. 5-(4-Chlorophenyl)methylidene-3-(2-hydroxyethyl)-
2-phenylimino-4-thiazolidinone (7d). Yield 62%; mp
1
140–142 °C; H NMR (CDCl3): d 4.00 (t, 2H, NCH2,
1
magnetic resonance spectrometer (300 MHz for H and
J = 4.2 Hz); 4.28 (m, 2H, CH2OH); 7.02–7.85 (m, 9H,
arom); 7.73 (s, 1H, CH); 13C NMR (CDCl3): d 46.1
(NCH2); 61.3 (CH2OH); 121.1, 125.6, 129.1, 129.5,
131.3 (CH arom); 121.9 (5-C); 130.7 (CH methylidene);
132.1, 136.0, 147.3, 152,2 (Cq); 167.0 (CO); Anal.
(C18H15ClN2OS) C, H, N.
75 MHz for 13C). Chemical shifts are given in d units
(ppm) relative to internal standard Me4Si and refer to
DMSO-d6 solutions. Coupling constants (J) are given in
hertz (Hz). 13C NMR spectra were determined by At-
tached Proton Test (APT) experiments and the reso-
nances were always attributed by proton–carbon
heteronuclear chemical shift correlation.
6.3.4. 3-(2-Hdroxyethyl)-5-(4-methoxyphenyl)methylidene-
2-phenylimino-4-thiazolidinone (7e). Yield 64%; mp 135–
1
Unless stated otherwise, all materials were obtained
from commercial suppliers and used without further
purification.
137 °C; H NMR (CDCl3): d 3.84 (s, 3H, OCH3); 4.00
(t, 2H, NCH2, J = 4.8 Hz); 4.30 (m, 2H, CH2OH);
6.90–7.43 (m, 9H, arom); 7.77 (s, 1H, CH); 13C NMR
(CDCl3): d 46.2 (NCH2); 55.5 (OCH3); 61.6 (CH2OH);
114.7, 121.7, 125.6, 129.3, 132.2 (CH arom); 117.8
(5-C); 131.9 (CH methylidene); 126.2, 146.6, 154.0,
161.2 (Cq); 167.1 (CO); Anal. (C19H18N2O2S) C, H, N.
6.2. Synthesis of 3-(3-hydroxypropyl)-2-phenylimino-4-
thiazolidinone (6)
Methyl bromoacetate (2.29 g, 15 mmol) and triethyl-
amine (2.02 g, 20 mmol) were added to a solution of
N-3-hydroxypropyl-N0-phenyl-thiourea (4) (10 mmol)
in ethanol (50 mL) and the mixture was refluxed under
stirring for 24 h. The crude mixture was treated with
ethyl ether until precipitation of triethylammonium bro-
mide, which was eliminated by filtering. The filtrate was
concentrated in vacuo and crystallized from ethanol.
Yield 85%; mp 143–145 °C; H NMR (CDCl3): d 1.92
(m, 2H, CH2); 3.65 (t, 2H, NCH2, J = 5.7 Hz); 3.86 (s,
2H, 5-CH2); 4.06 (m, 2H, CH2OH); 6.95–7.38 (m, 5H,
arom); Anal. (C12H14N2O2S) C, H, N.
6.3.5. 3-(3-Hydroxypropyl)-5-(4-methylthiophenyl)meth-
ylidene-2-phenylimino-4-thiazolidino-ne (8a). Yield 62%;
mp 120–122 °C; 1H NMR (CDCl3): d 2.00 (m, 2H,
CH2); 2.51 (s, 3H, SCH3); 3.69 (t, 2H, NCH2,
J = 5.4 Hz); 4.20 (m, 2H, CH2OH); 7.04–7.46 (m, 9H,
arom); 7.74 (s, 1H, CH); 13C NMR (CDCl3): d 14.9
(SCH3); 30.6 (CH2); 39.5 (NCH2); 58.3 (CH2OH);
119.7 (5-C); 121.3, 125.3, 126.0, 129.5, 130.3 (CH arom);
131.1 (CH methylidene); 129.7, 142.3, 147.4, 152.6 (Cq);
167.2 (CO); Anal. (C20H20N2O2S2) C, H, N.
1
6.3.6. 3-(3-Hydroxypropyl)-5-(3-methoxyphenyl)methyli-
dene-2-phenylimino-4-thiazolidinone (8c). Yield 65%; mp
90–92 °C; 1H NMR (CDCl3): d 2.00 (m, 2H, CH2); 3.68
(t, 2H, NCH2, J = 5.7 Hz); 3.81 (s, 3H, OCH3); 4.21 (m,
2H, CH2OH); 6.92–7.44 (m, 9H, arom); 7.76 (s, 1H,
CH); 13C NMR (CDCl3): d 30.6 (CH2); 39.6 (NCH2);
55.2 (OCH3); 58.6 (CH2OH); 115.3, 115.5, 121.1,
122.1, 125.2, 129.3, 131.5 (CH arom); 121.6 (5-C);
130.1 (CH methylidene); 134.8, 147.4, 152.4, 159,9
(Cq); 167.1 (CO); Anal. (C20H20N2O3S) C, H, N.
6.3. General procedures for the synthesis of 5-arylidene-3-
hydroxyalkyl-2-phenylimino-4-thiazolidinones 7–8a, c–e
The appropriate benzaldehyde (30 mmol) was added to
a solution of 5 or 6 compounds (30 mmol) and piperi-
dine (50 mmol) in ethanol (35 mL) and the mixture
was heated at reflux for 24 h. After this time, the solu-
tion was concentrated in vacuo and the residue was
recrystallized from methanol.
6.3.1. 3-(2-Hydroxyethyl)-5-(4-methylthiophenyl)methyli-
dene-2-phenylimino-4-thiazolidinone (7a). Yield 65%; mp
161–163 °C; H NMR (CDCl3): d 2.50 (s, 3H, SCH3);
6.3.7. 5-(4-Chlorophenyl)methylidene-3-(3-hydroxypropyl)-
2-phenylimino-4-thiazolidinone (8d). Yield 61%; mp
99–101 °C; H NMR (CDCl3): d 2.00 (m, 2H, CH2);
1
1
4.01 (t, 2H, NCH2, J = 4.2 Hz); 4.29 (m, 2H, CH2OH);
7.04–7.44 (m, 9H, arom); 7.74 (s, 1H, CH); 13C NMR
(CDCl3): d 15.0 (SCH3); 46.0 (NCH2); 61.5 (CH2OH);
120.0 (5-C); 121.4, 125.0, 125.7, 129.4, 130.3 (CH arom);
131.2 (CH methylidene); 129.7, 142.2, 147.4, 152.7 (Cq);
176.2 (CO); Anal. (C19H18N2O2S2) C, H, N.
3.68 (t, 2H, NCH2, J = 5.7 Hz); 4.21 (m, 2H, CH2OH);
7.02–7.45 (m, 9H, arom); 7.72 (s, 1H, CH); 13C NMR
(CDCl3): d 31.0 (CH2); 40.0 (NCH2); 58.7 (CH2OH);
121.5, 125.6, 129.7, 130.3, (CH arom); 122.2 (5-C);
131.4 (CH methylidene); 132.3, 136.3, 147.7, 152.2
(Cq); 167.2 (CO); Anal. (C19H17ClN2OS) C, H, N.
6.3.2. 3-(2-Hydroxyethyl)-5-(3-methoxyphenyl)methyli-
dene-2-phenylimino-4-thiazolidinone (7c). yield 65%; mp
91–93 °C; 1H NMR (CDCl3): d 3.82 (s, 3H, OCH3);
4.01 (t, 2H, NCH2, J = 4.8 Hz); 4.3 (t, 2H, CH2OH,
6.3.8. 3-(3-Hydroxypropyl)-5-(4-methoxyphenyl)methyli-
dene-2-phenylimino-4-thiazolidinone (8e). Yield 65%; mp
129–131 °C; H NMR (CDCl3): d 1.98 (m, 2H, CH2);
3.66 (t, 2H, NCH2, J = 5.7 Hz); 3.83 (s, 3H, OCH3);
1

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7624
4.20 (m, 2H, CH2OH); 6.92–7.42 (m, 9H, arom); 7.74 (s,
1H, CH); 13C NMR (CDCl3): d 30.9 (CH2); 39.4
(NCH2); 55.3 (OCH3); 58.4 (CH2OH); 114.7, 121.3,
125.9, 129.4, 131.9 (CH arom); 118.2 (5-C); 131.5 (CH
methylidene); 126.1, 147.6, 152.8, 161.0 (Cq); 167.4
(CO); Anal. (C20H20N2O2S) C, H, N.
cytes. This was washed 3 times with DMEM supple-
mented with 10% foetal calf serum (FCS), and was
subjected to vital colouration method staining with eo-
sin in order to determine the number and the vitality
of recovered cells. After 24 h the medium was removed
and cells were treated as follows: (a) Control, (b) IL-1b
(10 ng/mL), (c) Indomethacin (10ꢀ5 M) + IL (10 ng/mL),
d–h) 7a–7e (10, 50 and 100 lg/mL) + IL (10 ng/mL), i–m)
8a–8e (10, 50 and 100 lg/mL) + IL (10 ng/mL), n) 2 (10,
50 and 100 lg/mL) + IL (10 ng/mL).
6.4. Synthesis of 3-hydroxyalkyl-5-(4-methylsulfonylphe-
nyl)methylidene-2-phenylimino-4-thiazolidinone 7b, 8b
A suspension of compound 7a or 8a (0.2 mol) in 30 mL
acetic acid (50%) was treated dropwise with H2O2 35%
(0.3 mol, 30 g) and stirred for 48 h at room temperature.
The mixture was then filtered and the residue was trea-
ted dropwise with H2O2 35% (0.3 mol, 30 g) and stirred
for 48 h at room temperature. The residue was filtered
and recrystallized from methanol.
After 120 h the supernatants of cartilage culture were
collected for different assays.
7.1. Cell viability assay
The cytotoxic effect of the experimental substances was
evaluated by a cell viability test based on the cleavage
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) by mitochondrial dehydrogenases of
metabolically active cells.5,6,17
6.4.1. 3-(2-Hydroxyethyl)-5-(4-methylsulfonylphenyl)meth-
ylidene-2-phenylimino-4-thiazolidinone (7b). Yield 93%;
mp 208–210 °C; 1H NMR (CDCl3): d 3.01 (s, 3H,
SO2CH3); 3.9 (t, 2H, NCH2, J = 5.1 Hz); 4.00 (m, 2H,
CH2OH); 7.68–8.06 (m, 9H, arom); 7.93 (s, 1H, CH);
13C NMR (CDCl3): d 44.1 (SO2CH3); 46.3 (NCH2);
61.7 (CH2OH); 125.7 (5-C); 121.2, 125.0, 125.6, 128.0,
129.5 (CH arom); 130.3 (CH methylidene); 138.7,
141.0, 147.0, 151.8, (Cq); 167.2 (CO); Anal.
(C19H18N2O4S2) C, H, N.
7.2. Determination of nitrite levels
Nitrite was determined by adding 100 ll of Griess
reagent (1% sulfanylamide and 0.1% naphthyl-ethylene-
diamine dihydrochloride in 5% of hydrochloric acid) to
100 ll of samples.18 The optical density at k = 570 nm
was measured using a microtitre plate reader. Nitrite
concentrations were calculated by comparison with
respective optical densities of standard solutions of
sodium nitrite prepared in medium.
6.4.2. 3-(3-Hydroxypropyl)-5-(4-methylsulfonylphenyl)meth-
ylidene-2-phenylimino-4-thiazolidinone (8b). Yield 92%; mp
1
101–103 °C; H NMR (CDCl3): d 2.05 (m, 2H, CH2);
3.06 (s, 3H, SO2CH3); 3.68 (t, 2H, NCH2, J = 5.7 Hz);
4.21 (m, 2H, CH2OH); 7.01–7.99 (m, 9H, arom); 7.79
(s, 1H, CH); 13C NMR (CDCl3): d 30.5 (CH2); 39.9
(NCH2); 44.4 (SO2CH3); 58,4 (CH2OH); 125.5 (5-C);
121.2, 124.2, 125.5, 127.9, 129.5 (CH arom); 130.3
(CH methylidene); 138.7, 140.9, 147.1, 151.1 (Cq);
166.4 (CO); Anal. (C20H20N2O4S2) C, H, N.
7.3. Determination of glycosaminoglycans (GAGs)
The level of GAGs was measured by spectrophotometry
with a solution of 1.9-dimethylmethylene blue at
k = 535 nm.19 The amount of glycosaminoglycans was
calculated from a standard curve obtained for shark
chondroitin sulfate.
7. Cell isolation and human articular chondrocyte culture
7.4. Determination of prostaglandins (PGE2)
Human articular cartilage was obtained at replacement
surgery from patients with femoral neck fractures. The
isolation procedure was conducted under aseptic condi-
tions. The cartilage was cut into small fragments and
carefully washed using Dulbecco’s Modified Eagle’s
Medium (DMEM) culture medium containing NaH-
CO3, 25 mM Hepes, 1 mM sodium piruvate, 50 mg/mL
gentamycin, 100 U/mL penicillin, 100 mg/mL strepto-
mycin and 2.5 mg/mL amphotericin B. Chondrocytes
were isolated through 3 sequential passages of enzy-
matic 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; incubation with
0.5% pronase type XIV (5 mg/mL for 100 mg of carti-
lage), 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 cellular suspension obtained
was filtered (filters from 100 and 70 mm) to eliminate
the residues of the digestion and the cellular aggregates
and to obtain a monocellular suspension of chondro-
PGE2 was determined in the culture supernatant by the
enzyme immunoassay (EIA) system using a commer-
cially available immunoassay kit (Amersham Pharma-
cia, UK) according to the manufacturer’s instructions.
The detection limit is 1 pg/mL. The values were ex-
pressed as pg/mL PGE2 released.
7.5. Determination of MMP-3
MMP-3 was determined in the culture supernatant by
the ELISA system using a commercially available immu-
noassay kit (Amersham-Pharmacia, UK) according to
the manufacturer’s instructions. The detection limit is
1 ng/mL. The values were expressed as ng/mL MMP-3
released.
7.6. Statistical analysis
All the present results are means SEM of three exper-
iments performed on quadruplicate samples. The

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R. Ottana et al. / Bioorg. Med. Chem. 15 (2007) 7618–7625
7625
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8. Ottana, R.; Mazzon, E.; Dugo, L.; Monforte, F.; Maccari,
R.; Sautebin, L.; De Luca, G.; Vigorita, M. G.; Alcaro, S.;
Ortuso, F.; Caputi, A. P.; Cuzzocrea, S. Eur. J. Pharma-
col. 2002, 71, 448.
Student’s t-test was used to evaluate the differences be-
tween the means of each group. A value of P < 0.05
was considered to be statistically significant.
`
9. Ottana, R.; Maccari, R.; Barreca, M. L.; Bruno, G.;
Rotondo, A.; Rossi, A.; Chiricosta, G.; Di Paola, R.;
Sautebin, L.; Cuzzocrea, S.; Vigorita, M. G. Bioorg. Med.
Chem. 2005, 13, 4243.
Acknowledgement
This work was financially supported by the Fondo Ate-
neo di Ricerca (University of Messina, Italy).
10. Vicini, P.; Incerti, M.; Cardile, V.; Garufi, F.; Ronsisvalle,
S.; Panico, A. M. ChemMedChem 2007, 1, 113.
11. Emoto, K.; Futaki, K.; Tsukahara, H. DE Patent
2129087, 1971.
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