Heteroarylimino-4-thiazolidinones as inhibitors of cartilage degradation

Article abstract of DOI:10.1016/j.bioorg.2010.11.002

2-Benzo[d]thiazolyl- and 2-benzo[d]isothiazolyl-imino-5-benzylidene-4- thiazolidinone derivatives were investigated as potential metalloproteinases (MMPs) inhibitors and evaluated for their antidegenerative activity on human chondrocyte cultures stimulate

Full text of DOI:10.1016/j.bioorg.2010.11.002

Bioorganic Chemistry 39 (2011) 48–52
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Heteroarylimino-4-thiazolidinones as inhibitors of cartilage degradation
b,
c
b
d
a
Anna Maria Panico a, , Paola Vicini , Athina Geronikaki , Matteo Incerti , Venera Cardile , Lucia Crascì ,
⇑
⇑
Rossella Messina a, Simone Ronsisvalle a
a Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
b Pharmaceutical Department, Faculty of Pharmacy, University of Parma, V.le G.P. Usberti 27/A, 43124 Parma, Italy
c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
d Department of Physiological Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 October 2010
Available online 5 December 2010
2-Benzo[d]thiazolyl- and 2-benzo[d]isothiazolyl-imino-5-benzylidene-4-thiazolidinone derivatives were
investigated as potential metalloproteinases (MMPs) inhibitors and evaluated for their antidegenerative
activity on human chondrocyte cultures stimulated by IL-1b, using an experimental model that repro-
duces the mechanisms involved in osteoarthritic (OA) diseases. Cell viability, the amount of glycosami-
noglycans (GAGs) and the production of nitric oxide (NO) were measured. The most potent compound,
5-(4-methoxy-benzylidene)-2-(benzo[d]isothiazol-3-ylimino)-thiazolidin-4-one (4b), a MMP-13 inhibi-
Keywords:
Heteroarylimino-4-thiazolidinones
Metalloproteinases
Osteoarthritis
Nitric oxide
Glycosaminoglycans
tor at nanomolar concentration (IC50 = 0.036
opment of novel clinical agents, inhibitors of cartilage degradation, for the treatment of OA.
Ó 2010 Elsevier Inc. All rights reserved.
lM), could be considered as a lead compound for the devel-
1. Introduction
endopeptidases collectively capable of degrading all ECM compo-
nents, in pathological situations as OA.
The matrix metalloproteinases (MMPs) are a family of zinc
endopeptidases that have been implicated in a wide variety of bio-
logical processes and pathological conditions such as cancer, heart
failure, atherosclerosis and arthritis [1–4]. Increased levels of these
enzymes, that are responsible for degradation of aggrecan and
collagen, have been observed in the cartilage of patients with
osteoarthritis (OA) and correlate with the severity of the disease.
OA is a multifactorial disease involving a progressive destruc-
tion of joint tissue and cartilage through a complex series of
mechanisms. Cartilage consists of a relatively small number of
chondrocytes and many extracellular matrix (ECM) components,
comprising mainly collagen fibrils and aggrecan (a large aggregat-
ing proteoglycan). Chondrocytes synthesize and catabolise ECM
macromolecules, while the matrix in turn maintains the homeosta-
sis of the cellular environment and cartilage structure. In OA dis-
eases, the degradation of the ECM exceeds its synthesis, resulting
in a net decrease in the amount of cartilage matrix. The deregu-
lated synthesis of matrix components involves key molecules in
the erosion of cartilage, such as proteoglycan, aggrecan and type
II collagen, whose loss and destruction have recently been recog-
nized as significant factors in OA [5]. The primary cause of this pro-
cess is an increase in the activities of the matrix metalloproteinases
Currently, approximately 27 MMPs are known, grouped into
subfamilies of collagenases, gelatinases, stromelysins, membrane-
type MMPs, and novel MMPs, on the basis of their structure and
substrate specificity [6–8]. The main MMPs involved in the patho-
genesis of OA are collagenases, stromelysins and gelatinases with
the ability to degrade triple helix collagen, proteoglycans and
denatured collagen, respectively [9]. Stromelysins are recognized
as collagenase-related connective tissue-degrading metallopro-
teinases capable of degrading ECM multiple components, or
stroma. Stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10)
isoforms are similar in their amino acid sequence and substrate
specificity. MMP-3, stromelysin-1, is particularly destructive to-
wards matrix components including the protein structure of prote-
oglycans [10,11]. MMP-13, collagenase, consistently found in the
deeper layers appears to be the major mediator of type II collagen
destruction [12–14]. Members of the collagenase subgroup of
MMPs, i.e. collagenase-1 (MMP-1), collagenase-2 (MMP-8), and
collagenase-3 (MMP-13), are the principal neutral proteinases
capable of degrading native fibrillar collagens in the extracellular
space. They all cleave types I, II, and III collagens; MMP-13 cleaves
fibrillar collagens with preference to type II collagen over types I
and III collagens.
(MMPs),
a
family of structurally related zinc-dependent
In addition, the functional alteration of cartilage also results
from the intervention of different cytokines, especially IL-1b. It in-
duces high levels of pro-inflammatory mediators, such as prosta-
glandins E2 (PGE2) and nitric oxide (NO), and inhibits collagen
and proteoglycan synthesis. IL-1b is a pivotal driving force in
⇑
Corresponding authors. Fax: +39 095 7384270 (A.M. Panico), +39 0521 905006
(P. Vicini).
E-mail addresses: panico@unict.it (A.M. Panico), paola.vicini@unipr.it (P. Vicini).
0045-2068/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.11.002

A.M. Panico et al. / Bioorganic Chemistry 39 (2011) 48–52
49
inducing and sustaining cartilage damage that usually leads to the
loss of sulfated glycosaminoglycans (GAGs) [15]. GAG release is a
consequence of increased matrix protease activity, leading to the
cleavage of collagen and proteoglycans. High levels of PGE2 also
mediate cartilage resorption by decreasing chondrocyte prolifera-
tion, enhancing MMPs activity and inhibiting aggrecan synthesis
in chondrocytes [16]. During inflammatory processes, that repre-
sent one of the key pathogenetic aspects of degenerative joint dis-
ease, PGE2 production is intensified and this contributes to
synovial inflammation by increasing local blood flow and by poten-
tiating the effects of mediators such as bradykinin, responsible for
relevant vasopermeability. It has been shown that PGE2 is able to in-
hibit chondrocyte growth and upregulate IL-1b [17]. The above find-
ings demonstrate the importance of these mediators in human OA.
The pharmacological formulary currently possesses few weap-
ons against joint destruction [18,19] indicating the need for devel-
opment of molecules capable of such inhibitory activity. As a part
of an intensive research program focused on the design and syn-
thesis of new chondroprotective/anti-inflammatory drugs [20,21],
our interest in this field prompted us to study 4-thiazolidinone
derivatives 1a–6a and 1b–6b (Table 1) belonging to a chemical
class of molecules, the 4-thiazolidinones, that have been demon-
strated to possess anti-inflammatory properties and a protective
action on inflammatory degenerative diseases [22–24], such as OA.
We here report the MMP-3 and MMP-13 inhibition capability
(IC50) and the antidegenerative/chondroprotective effect, determined
by evaluating cell viability, NO and GAGs levels of 2-(benzo[d]-
thiazol-2-ylimino)-thiazolidin-4-one (1a), 2-(benzo[d]thiazol-2-
ylimino)-5-benzylidenethiazolidin-4-ones (2a–6a) and of their
isomers 2-(benzo[d]isothiazol-3-ylimino)-thiazolidin-4-one (1b)
and 2-(benzo[d]isothiazol-3-ylimino)-5-benzylidenethiazolidin-4-
ones (2b–6b). In order to find the pharmacophore and explore the
structure–activity relationships of these compounds we considered
simple substitutions of the benzylidene moiety based on previous
SAR studies.
2. Materials and methods
2.1. Chemistry
The general synthetic approach to heteroarylimino-4-thiazolid-
inones is shown in Fig. 1. The compounds described here (Table 1)
were synthesized by the multi-step reaction protocol reported ear-
lier by us [25].
2.2. MMP-3 and 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 per-
formed in 50 mM HEPES buffer for MMP-13, MES 50 mM only for
Table 1
MMP-3 and MMP-13 inhibitory activity of compounds synthesized.
1a-6a
H
N
O
R
N
S
N
S
1b-6b
R-
H
Comp
MMP-3, IC50
(lM)
MMP-13, IC50
(lM)
Comp
MMP-3, IC50
(
lM)
MMP-13, IC50 (lM)
n.a.a
32
1a
2a
36.68
44
n.a.a
45
1b
2b
n.a.a
n.a.a
3a
4a
5a
6a
n.a.a
16.7
65
n.a.a
98
3b
4b
5b
6b
10.35
n.a.a
65
0.158
0.036
25
20
n.a.a
60
n.a.a
55.88
Representative results of at least three independent experiments are reported.
a
Not active: IC50 (lM) > 100.

50
A.M. Panico et al. / Bioorganic Chemistry 39 (2011) 48–52
Fig. 1. General synthetic scheme.
MMP-3, containing 5 mM CaCl2, 0.1 mM ZnCl2, 0.05% Brij-35, at pH
7, using 10 nM of proteolytic enzyme (catalytic domains of MMP-3
and MMP-13) (Biomol, Inc.) and 350 nM of peptide. The enzyme
was incubated at 25 °C with increasing concentration of the inhib-
itor and the fluorescence (excitationmax 328 nm; emissionmax
393 nm) was measured for 3 min after the addition of the substrate
using a Varian Eclipse fluorimeter. Fitting of rates as a function of
inhibitor concentration provided IC50 values. The inhibitor N-iso-
butyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (Bio-
mol, Inc.) was used as control [26].
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by
mitochondrial dehydrogenases of metabolically active cells [27].
2.5. Determination of nitrite
Nitrite was determined by adding 100
sulphanylamide and 0.1% naphthylethylenediamine dihydrochlo-
ride in 5% of hydrochloric acid) to 100 L of samples [28]. The opti-
lL of Griess reagent (1%
l
cal density at k = 570 nm was measured using a microtiter plate
reader. Nitrite concentrations were calculated by comparison with
respective optical densities of standard solutions of sodium nitrite
standard curve (0–120 lM).
2.3. Cell isolation and human articular chondrocyte culture
2.6. Determination of glycosaminoglycans
Normal human articular cartilage was obtained at replacement
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 Mod-
ified Eagles Medium (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
amphotericin B. Chondrocytes were isolated through three sequen-
tial 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; incubation 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 filtered (filters from 100 and 70 mm) to eliminate the residues
of the digestion and cellular aggregates. Freshly isolated chondro-
cytes were seeded into monolayer culture at a cell density of
2 Â 105 cells and cultured in 1 mL of DMEM supplemented with
10% fetal 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, appropri-
ately diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) and
dispensed to the wells to give to different concentrations, from
The level of GAGs, an index of cartilage damage, was measured
by spectrophotometry with a solution of 1,9-dimethylmethylene
blue at k = 535 nm using the method of Farndale et al. [29]. The
amount of glycosaminoglycans was calculated from a standard
curve (100–500 lg/mL) obtained for shark chondroitin sulfate C,
derived from shark cartilage.
2.7. Statistical analysis
All the present results are means SEM of three experiments
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 statisti-
cal 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 soft-
ware package SYSTAT, version 9 (Systat Inc., Evanston IL, USA).
3. Results and discussion
As shown in Table 1, many of the tested compounds displayed
inhibitory activity (IC50) against MMP-3 and MMP-13. The MMP as-
says were performed evaluating the ability of compounds 1a–6a
and 1b–6b to prevent the hydrolysis of the fluorescence-quenched
peptide substrate Mca-Pro-Gly-Leu-Dpa-Ala-Arg-NH2. Marked
differences in potency and selectivity were observed for the analogs
with different core structures (benzo[d]thiazol-3-ylimino or benzo-
[d]isothiazol-3-ylimino). The highest activity and selectivity for
MMP-13 was exhibited by the group of 2-(benzo[d]isothiazol-3-
ylimino)-5-benzylidenethiazolidin-4-ones (2b–6b). The 5-(4-hydro-
xybenzylidene)-2-(benzo[d]isothiazol-2-ylimino)thiazolidin-4-one
10 lg/mL to 0.01 lg/mL. IL-1b was used at 10 ng/mL alone or com-
bined with experimental compounds. The assays were performed
after 120 h of treatment.
2.4. 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-

A.M. Panico et al. / Bioorganic Chemistry 39 (2011) 48–52
51
(3b) and 5-(4-methoxybenzylidene)-2-(benzo[d]isothiazol-2-yli-
mino)thiazolidin-4-one (4b) derivatives resulted the most potent
MMP-13 inhibitors with IC50 value of 0.158 and 0.036 M, respec-
tively. The structure–activity relationship analysis of the data
reported in Table 1 shows also that the parent compounds were
inactive (1a against MMP-13 and 1b against both the enzymes:
(IC50 60 and 55.88
l
M) over MMP-3 (IC50 > 100
l
M). This suggests
that the electronic and hydrophilic/lipophilic effects of the substit-
uents on the benzene ring plays an important role in the modulation
of MMPs inhibitory activity and hydrophilic, electron-donating
groups seem to favor the potency and the selectivity as shown by
compounds 3b and 4b against MMP-13.
l
IC50 > 100
lM), or moderately active (1a for MMP-3, IC50
=
The compounds were in vitro assayed on chondrocytes at differ-
39.68 M), suggesting that the unsubstituted and substituted
l
ent concentrations from 10
lg/mL to 0.01 lg/mL. The results are
5-arylidene moiety plays an important role in enhancing the inhib-
itory activity of MMPs. Among the 5-arylidene derivatives, the
inhibitory effect appears to be dependent on the substitution at
the benzene ring. The introduction of the hydrophilic, electron-
donating hydroxy group at the para position of the benzylidene
moiety (3b) increased, in the benzisothiazolylimino class, the inhib-
itory potency against MMP-13 and MMP-3 with IC50 of 10.35 and
reported at 10 g/mL, representing the best data for cellular assays,
l
probably due to the different behavior of the compounds on bio-
logical systems. The tetrazolium dye, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), fluorogenic assay
was chosen as the method for quantifying cell viability. All tested
compounds did not reduce the ability of chondrocytes to metabo-
lise tetrazolium salts, demonstrating that they did not interfere
with cell viability (data not shown).
Fig. 2 shows NO production levels by IL-1b stimulated articular
chondrocytes 120 h after the addition of compounds 1a–6a, and
their isomers 1b–6b at a concentration of 10 lg/mL. The tested
compounds strongly inhibited NO production induced by
inflammatory IL-1b. The 4-chlorosubstituted derivatives (5a, 5b)
and the 4-nitrosubstituted compounds (6a, 6b) of both benzo-
thiazolylimino and benzisothiazolylimino series, when combined
with IL-1b, showed a significant reduction in NO release, compared
0.158
(3a) did not show inhibition, for both MMP-3 and MMP-13, at or
below 100 M. The introduction of the moderately hydrophilic,
electron-donating methoxy group at the benzylidene para position
in the benzisothiazolylimino analog (4b) resulted in IC50 > 100
against MMP-3 and in the highest increase in activity on MMP-13
(IC50 = 0.036 M); notably, its benzothiazolylimino isomer (4a)
displayed a sixfold selectivity for MMP-3 over MMP-13 (IC50
16.7 versus 98 M). The introduction of the lipophilic/electron-
lM, respectively. In contrast, the benzothiazolylimino isomer
l
lM
l
l
withdrawing chloro group in the isomeric compounds 5a and 5b,
afforded increase of potency, against MMP-13 with respect to the
unsubstituted analogs, as well as against MMP-3 for 5b, while a
slight reduction of activity was observed in the activity of 5a on
MMP-3. A similar effect was observed between the isomeric com-
pounds 6a and 6b bearing the nitro electron-withdrawing group
at the benzylidene para position; with respect to their chloro
analogs they showed a significant enzyme selectivity for MMP-13
to the sample treated only with IL-1b (21.68
compound 5a proved to be the most efficacious derivative
(4.87 M), as well as its isomer 5b (9.55 M). Furthermore, com-
pound 6b exhibited a level of NO production of 5.18 M, while
its isomer (6a) showed a level of NO production of 9.15 M. This
indicates that insertion of lipophilic, electron-withdrawing groups
at the benzylidene para position is the most favorable substitution
to decrease the NO production levels. It is worth noting that the
lM). In particular,
l
l
l
l
Fig. 2. Effect of heteroarylimino-4-thiazolidinones on NO release. NO production (
l
M) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes
120 h after the addition of compounds 1a–6a and their isomers 1b–6b at 10 l
g/mL. ⁄P < 0.005.
Fig. 3. Effect of heteroarylimino-4-thiazolidinones on GAGs release. GAGs release (
lg/mL) (means SEM) in the culture medium by IL-1b stimulated articular chondrocytes
120 h after the addition of compounds 1a–6a and their isomers 1b–6b at 10
l
g/mL. ⁄P < 0.005.

52
A.M. Panico et al. / Bioorganic Chemistry 39 (2011) 48–52
parent compound 1a and its isomer 1b also exhibited good anti-
degenerative/anti-inflammatory activity being able to reduce the
NO levels induced by the pro-inflammatory IL-1b. It is noteworthy
that all the compounds inhibited the NO production induced
by IL-1b.
The level of GAGs was measured by the spectrophotometric
method of Farndale et al. that represents an index of cartilage dam-
age in chondrocyte cultures [30,31]. As can be seen in Fig. 3, all
compounds 1a–6a, and their isomers 1b–6b, combined with IL-
no)-thiazolidin-4-one (4b), a MMP-13 inhibitor at nanomolar
concentration (IC50 0.036 M) could be considered a lead com-
pound for the development of novel agents, inhibitors of cartilage
degradation, for the treatment of OA. Further exploration of the
SAR of these novel MMP inhibitors is under evaluation and will
be reported in due course.
l
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Two isomeric classes of heteroarylimino-4-thiazolidinones have
been explored and were found to be inhibitors of MMPs and degen-
erative/inflammation mediators, such as NO and GAGs. The novelty
of the 4-thiazolidinone scaffold for the MMPs activity represents
the main finding of the present study and the most potent com-
pound, 5-(4-methoxy-benzylidene)-2-(benzo[d]isothiazol-3-ylimi-
[31] M. Del Carlo, D. Schwartz, E.A. Erickson, R.F. Loeser, Free Radical Biol. Med. 42
(2007) 1350–1358.