Synthesis and anti-inflammatory activity of new arylidene-thiazolidine-2,4-diones as PPARγ ligands

Article abstract of DOI:10.1016/j.bmc.2010.04.045

Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known agonist of PPARγ, which was used as a reference drug. This suggests that the substituted 5-arylidene and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of compounds. Docking studies with these compounds indicated that they exhibit specific interactions with key residues located in the site of the PPARγ structure, which corroborates the hypothesis that these molecules are potential ligands of PPARγ. In addition, competition binding assays showed that four of these compounds bound directly to the ligand-binding domain of PPARγ, with reduced affinity when compared to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory activities of this set of molecules. The analysis of the docking results, which takes into account the hydrophilic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-di one compound had the best activity and the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substituted 5-arylidene group of this compound and the residues of the binding site.

Full text of DOI:10.1016/j.bmc.2010.04.045

Bioorganic & Medicinal Chemistry 18 (2010) 3805–3811
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-inflammatory activity of new
arylidene-thiazolidine-2,4-diones as PPAR ligands
c
Cleiton Diniz Barros a, , Angélica Amorim Amato b, Tiago Bento de Oliveira a, , Karime Bicas Rocha Iannini b,
Anekécia Lauro da Silva a, , Teresinha Gonçalves da Silva a, , Elisa Soares Leite c, Marcelo Zaldini Hernandes c,
Maria do Carmo Alves de Lima a, , Suely Lins Galdino a, , Francisco de Assis Rocha Neves b,
Ivan da Rocha Pitta a, ,
*
a Laboratório de Planejamento e Síntese de Fármacos—LPSF, Grupo de Pesquisa em Inovação Terapêutica—GPIT, Universidade Federal de Pernambuco,
Avenida Moraes Rego 1235, Cidade Universitária, CEP 50670-901 Recife, Pernambuco, Brazil
b Laboratório de Farmacologia Molecular, Departamento de Ciências Farmacêuticas, Faculdade de Ciências da Saúde, Universidade de Brasília, Brazil
c Laboratório de Química Teórica Medicinal—LQTM, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight new 5-arylidene-3-benzyl-thiazolidine-2,4-diones with halide groups on their benzyl rings were
synthesized and assayed in vivo to investigate their anti-inflammatory activities. These compounds
showed considerable biological efficacy when compared to rosiglitazone, a potent and well-known
agonist of PPARc, which was used as a reference drug. This suggests that the substituted 5-arylidene
and 3-benzylidene groups play important roles in the anti-inflammatory properties of this class of com-
Received 24 February 2010
Revised 14 April 2010
Accepted 16 April 2010
Available online 21 April 2010
pounds. Docking studies with these compounds indicated that they exhibit specific interactions with key
Keywords:
Arylidene-thiazolidinedione
Anti-inflammatory activity
residues located in the site of the PPAR
cules are potential ligands of PPAR . In addition, competition binding assays showed that four of these
compounds bound directly to the ligand-binding domain of PPAR , with reduced affinity when compared
c structure, which corroborates the hypothesis that these mole-
c
c
PPAR
c
to rosiglitazone. An important trend was observed between the docking scores and the anti-inflammatory
activities of this set of molecules. The analysis of the docking results, which takes into account the hydro-
philic and hydrophobic interactions between the ligands and the target, explained why the 3-(2-bromo-
benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-dione compound had the best activity and
the best docking score. Almost all of the stronger hydrophilic interactions occurred between the substi-
tuted 5-arylidene group of this compound and the residues of the binding site.
Docking
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
activity was thought to be limited to lipid metabolism and glucose
homeostasis. Later studies showed that PPAR activation regulates
The correct function of a tissue is indispensable for the proper
function of the body. When tissues are injured through physical
damage or are infected by exogenous microbial organisms, local
and systemic responses are activated with the primary goals of elim-
inating the offending factors as fast as possible, restoring the tissue
integrity, and retaining information about the offending agent to
facilitate recognition and elimination on a future encounter. The
outcome of these responses is a rapid physiological response of
the body to damage and infection, that is, inflammation.1
inflammatory responses, cell proliferation and differentiation, as
well as apoptosis.2,3 The involvement of PPAR
in inflammatory pro-
cesses was first suggested by the antagonism between the activities
of proinflammatory cytokines and PPAR . Additionally, macrophage
activation is inhibited by several PPAR
agonists.4 Therefore, this
receptor is an attractive target for the development of anti-inflam-
matory agents due to its key roles at various stages in the inflamma-
tory process.
c
c
c
Thiazolidine-2,4-dione activates PPARc and is used as an antidi-
Peroxisome proliferator-activated receptors (PPARs) are mem-
bers of the nuclear hormone receptor superfamily which are li-
gand-activated transcription factors. So far, three PPAR isotypes
abetic drug in the treatment of type 2 diabetes.5,6 Thiazolidines,
such as rosiglitazone and pioglitazone (Fig. 1), are synthetic com-
pounds that bind and activate PPARc. The two thiazolidines share
have been reported: PPAR
a
, PPARb, and PPAR
c. Originally, PPAR
a common thiazolidine-2,4-dione structure that is responsible for
the majority of their pharmacological effects, including anti-
inflammatory effects.7,8
Recently, arylidene-thiazolidinediones were evaluated in the al-
loxan-induced hyperglycemia mice model, where the biomolecular
* Corresponding author. Tel./fax: +55 81 2126 8347.
E-mail address: irpitta@gmail.com (I.R. Pitta).
http://www.ufpe.br/gpit.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.045

3806
C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
H
O
H
O
N
N
N
N
O
O
S
O
O
N
S
Rosiglitazone
Pioglitazone
Figure 1. Rosiglitazone and pioglitazone structures that bind PPARc with a high affinity.
target considered to be responsible for the process was nuclear
PPARc 9
The present work describes the synthesis, anti-inflamma-
mine atom in position 2 of the benzyl ring. On the other hand, com-
pounds 5 and 8, which have two substituents in the benzyl ring,
only showed moderate activity when compared to rosiglitazone.
After optimization of the arylidene-thiazolidinediones using the
AM1 method,12 agreement was observed with the previous results
obtained by Leite et al.,9 confirming that the Z isomer is the most
stable for all of the compounds.
The docking solutions (poses) of the arylidene-thiazolidinedi-
one compounds in the PPARc structure were compared to the po-
sition of the rosiglitazone docked in this receptor, as shown in
Figure 2. The docking solutions for the eight ligands were posi-
.
tory activity and structural characteristics of compounds derived
from the thiazolidine-2,4-diones substituted with an arylidene
and a benzyl group, as shown in Scheme 1. The arylidene was in-
cluded at position 5 with the substituents Br, Cl, OCH3, SO2CH3,
and C6H5, and the benzyl was included at position 3 with the sub-
stituents Cl, F, and Br. These selected substituents have been the
main subject of previous SAR investigations on thiazolidinones,
which were based upon the structural similarity between these
molecules and the Coxib derivatives.10,11 The effects of these substi-
tutions on the biological response were analyzed and docking stud-
ies were performed to investigate the binding patterns with the
tioned within the well-characterized site of the PPARc structure.
A closer examination of the interactions of these ligands with the
site shows that some key residues are involved in important
hydrophilic interactions (hydrogen bonds) with the arylidene-thia-
zolidinediones and with rosiglitazone. This can be seen in Figure 3,
where compound 11 that had the best activity and the best dock-
ing score in the series (À23.45 kJ molÀ1) was superposed with the
co-crystallized rosiglitazone in the presence of important residues
of the site. It is important to note that compound 6 has essentially
the same binding mode in comparison to 11 and presents the same
interactions, which is confirmed by the very similar docking scores
in Table 1, which were À22.83 and À23.45 kJ molÀ1 for 6 and 11,
respectively.
PPARc structure as a tool to support the hypothesis that these com-
pounds are anti-inflammatory agents and act as PPARc ligands.
2. Results and discussion
The arylidene-thiazolidinediones 5–12 (Scheme 1) were tested
for anti-inflammatory activity in an air-pouch assay in which they
were evaluated for the ability to inhibit leukocyte migration from
blood circulation, as illustrated in Table 1. The arylidene-thiazolid-
inediones 6, 7, 9 and 11 were the most active anti-inflammatory
agents; in particular, ligand 11 had an anti-inflammatory activity
of 73.3%, which was slightly higher than that rosiglitazone
(72.0%). It should be noted that 6, 7 and 9 contain a chlorine atom
at position 3 of the benzyl ring and compound 11 contains a bro-
In Table 2, the hydrophilic and hydrophobic interactions be-
tween the ligands 11, 12 and rosiglitazone and the residues of
the PPAR
c
site are listed. It is possible to remark that ligand 11
H
O
O
O
N
N
N
benzyl halides
O
NaOH EtOH
R2CH=(CN)COOEt
Piperidine EtOH
H
S
S
O
S
O
R1
R1
R2
1
2, 3 or 4
5 to 12
Compound
R1
R2
5
6
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
2-Br
5-Br, 2-OCH3C6H3
4-SO2CH3C6H4
4-C6H5C6H4
7
8
3-Br, 4-OCH3C6H3
2-Fluorene
9
10
11
12
4-OCH3C6H4
4-SO2CH3C6H4
2,4-Cl2C6H3
2-Cl, 6-F
Scheme 1. Synthetic route for 5-arylidene-3-benzyl-thiazolidine-2,4-diones.

C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
3807
Table 1
Oral anti-inflammatory activity of the eight arylidene-thiazolidinediones using the air-pouch model and the respective docking scores
Compound
R1
R2
Dose (mg/kg)
PMNL counta (105/mL)
% Inhibitionb (PMNL)
Docking score (kJ molÀ1
)
5
6
7
8
9
10
11
12
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
2-Br
2-Cl, 6-F
—
5-Br, 2-OCH3C6H3
4-SO2CH3C6H4
4-C6H5C6H4
3-Br, 4-OCH3C6H3
2-Fluorene
4-OCH3C6H4
4-SO2CH3C6H4
2,4-Cl2C6H3
—
3
3
3
3
3
3
3
3
3
—
20.1 2.7
13.8 1.8
12.6 0.7
18.6 1.2
13.3 1.8
22.2 1.3
10.2 1.7
22.4 1.8
10.7 2.0
38.3 2.1
47.4
63.9
67.0
51.5
65.2
42.0
73.3
41.6
72.0
—
À9.99
À22.83
À19.72
À16.70
À18.54
À18.82
À23.45
À13.98
À34.03
—
Rosiglitazone
Control
—
—
a
PMNL is the cell infiltration by polymorphonuclear leukocytes. Data points represent the mean PMNL count number per animal group with the corresponding standard
error. The presented values are significant for the 95% confidence interval (ANOVA, Bonferroni test).
b
The inhibition was determined compared to a control group (untreated).
Figure 3. Docking results for the 3-(2-bromo-benzyl)-5-(4-methanesulfonyl-ben-
zylidene)-thiazolidine-2,4-dione (11; stick model in orange) and the co-crystallized
Figure 2. Superposition of the docking solutions for the eight arylidene-thiazolid-
inediones (5–12—Scheme 1) (line models in several colors) and the rosiglitazone
rosiglitazone (stick model in blue) interacting with important labeled residues of
(stick model in red) for the PPAR
c receptor (cartoon model). The co-crystallized
the PPARc receptor site. Compound 6, not shown in this figure for clarity reasons,
(experimental) rosiglitazone is also represented (stick model in blue). Figures 2 and
3 were generated using PyMOL v0.99.19
had essentially the same binding mode as 11, which can be confirmed by the similar
docking scores in Table 1, À22.83 and À23.45 kJ molÀ1 for 6 and 11, respectively.
ues in kJ molÀ1) of the complex between the ligand and the PPAR
receptor is related to greater anti-inflammatory activity or the per-
cent of inhibition of the PMNL count. This important result corrob-
c
(best activity) had many more interactions with the target then li-
gand 12 (worst activity). This explains, at a molecular level, why li-
gand 11 had a high docking score while ligand 12 had a low
docking score (À13.98 kJ molÀ1). The interactions with the co-crys-
tallized rosiglitazone were also included for comparison.
orates the hypothesis that these molecules act through the PPAR
c
target.
To determine whether the study compounds bind to the ligand-
binding domain of human PPAR (LBD-PPAR ), we assessed the
ability of saturated solutions of the compounds in DMSO to
displace [3H]rosiglitazone bound to His-LBD-hPPAR
. As shown
In Figure 4, the anti-inflammatory activity measured as the per-
cent of inhibition (using the PMNL count) and the docking scores
were plotted together, which exhibited an important trend. This
means that greater stability (the most negative docking score val-
c
c
c
Table 2
Hydrophilic and hydrophobic interactions between the docked ligands 11 and 12 and the co-crystallized rosiglitazone with respect to the residues of the PPAR
emphasized (bold format and ) in this table are the same interactions observed with the co-crystallized rosiglitazone
c
site. Residues
*
Co-crystallized rosiglitazone
Hydrophilic Hydrophobic
Ligand 11 (best activity)
Hydrophilic Hydrophobic
Ligand 12 (worst activity)
Hydrophilic
Hydrophobic
*
*
*
*
*
*
GLN286
ILE281
ARG288
CYS285
GLU291
GLU343
CYS285
GLY284
*
*
*
*
*
*
*
*
*
*
*
SER289
GLY284
CYS285
SER289
HIS323
ARG288
*
*
*
*
*
*
*
HIS323
HIS449
TYR473
SER289
LEU330
LEU333
ARG288
SER342
GLU343
GLN286
SER289
TYR327
*
*
*
HIS449
TYR473
*
*
*
*
*
ILE341
ILE341
MET348
HIS449
SER342
GLU343
HIS449
*
*

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C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
labeled rosiglitazone was observed for 6, 7, 11, and 12 with Ki val-
ues of 43 nM, 43.6 M, 7.3 M, and 4.0 M, respectively (Fig. 5B).
l
l
l
Taken together, these data indicate that 6, 7, 11, and 12 have the
ability to directly interact with the ligand-binding domain of
PPARc and are low affinity ligands for the receptor. We cannot rule
out the possibility that these compounds bind to the other PPAR
isotypes, since it has been shown that other thiazolidinediones,
including pioglitazone13 and rosiglitazone,14 bind and act as partial
agonists for PPARa and -d.
3. Conclusion
In summary, arylidene-thiazolidinediones were synthesized
and tested for their anti-inflammatory activities, and 3-(2-bro-
mo-benzyl)-5-(4-methanesulfonyl-benzylidene)-thiazolidine-2,4-
dione, compound 11, showed higher activity than the rosiglitazone
reference drug. The binding patterns observed for the docked com-
pounds strongly support the idea that they are possible ligands of
Figure 4. Trend between the docking results of the eight arylidene-thiazolidined-
the PPAR
LBD-PPAR
hPPAR in vitro and are low affinity ligands for the receptor.
Although compound 11 was shown to have higher anti-inflamma-
tory activity than rosiglitazone, it bound PPAR with 200-fold low-
er affinity than this reference ligand. In fact, previous studies have
c
c
receptor. Competition binding assays using purified His-
confirmed that 6, 7, 11 and 12 directly bind to LBD-
iones (5–12—Scheme 1) and rosiglitazone ligands in the PPAR
anti-inflammatory activities measured as the percent of inhibition with respect to
the PMNL count.
c receptor and the
c
c
in Figure 5A, unlabeled 6, 7, 11, and 12 but not 9 bound directly to
His-LBD-hPPAR
c. A concentration-dependent dissociation of radio-
characterized low affinity ligands for PPARc with potent insulin-
A
12000
10000
8000
6000
*
*
*
*
4000
2000
0
*
Veh
Rosiglitazone
6
11
12
7
9
10 µM
unlabeled arylidene thiazolidinediones (100 µM)
B
120
110
100
90
80
70
60
50
40
30
20
10
0
6 (K
i
i
43 nM)
7 (K
43,6 µM)
11 (K
i
i
7,3 µM)
4,0 µM)
12 (K
rosiglitazone (Ki1 5,3 nM)
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
log[compound] M
Figure 5. Competition of unlabeled arylidene-thiazolidinediones 6, 7, 9, 11, and 12 for the ligand-binding domain of PPAR
binding assay using a saturating concentration of unlabeled rosiglitazone (10 M) and the highest concentration at which the study compounds were soluble in DMSO
c
bound to [3H]rosiglitazone. (A) Competition
l
(100
l
M) revealed that arylidene-thiazolidinediones 6, 7, 11, and 12 displaced [3H]rosiglitazone from LBD-PPAR
c
.
(B) Concentration-dependent displacement of
*
[3H]rosiglitazone by unlabeled rosiglitazone and arylidene-thiazolidinediones 6, 7, 11 and 12. Values are expressed as the means SEM. Significantly different (p <0.05) from
vehicle control using one-way analysis of variance. Data are representative of at least two independent experiments.

C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
3809
sensitizing activity.15,16 Since improvement of insulin resistance by
PPAR agonist has been related to the anti-inflammatory effects of
the activated receptor,17 it is possible that the anti-inflammatory
activity of PPAR ligands is not directly correlated to their affinity
for the receptor.
The arylidene-thiazolidinedione compound 9, however, was not
characterized as a PPAR ligand by the competition binding assay,
although it had active anti-inflammatory properties in vivo, and
the results of the docking studies suggested that it interacts with
key residues in the site of the receptor.
ethanol–water mixture. The published chemical data on ethyl-3-
(5-bromo-2-methoxi-phenyl)-2-cyanoacrylate, ethyl-3-biphenyl-
c
4-yl-2-cyanoacrylate,
ethyl-3-(3-bromo-4-methoxi-phenyl)-2-
c
cyanoacrylate, ethyl-3-(4-methoxi-phenyl)-2-cyanoacrylate,26 and
ethyl-3-(2,4-dichloro-phenyl)-2-cyanoacrylate27 are not reported
here.
c
5-Arylidene-3-benzyl-thiazolidine-2,4-diones (5–12) general
procedure: An equimolar (0.83 mMol) mixture of 3-benzyl-thiazol-
idine-2,4-dione 2–4 and (2-cyano-3-phenyl)-ethyl acrylates dis-
solved in ethanol (10 mL) with piperidine (250 lL) was heated at
Additionally, an important in vivo versus in silico trend be-
tween the anti-inflammatory activities and the docking scores
was found, showing the appropriate choice of docking model used
for this study. This trend corroborates the hypothesis that arylid-
ene-thiazolidinediones act against the inflammation response
50 °C for 2–3 h. After cooling, the precipitated product was recrys-
tallized from an ethanol–water mixture or purified by flash column
chromatography. The melting points were measured in a capillary
tube on a Buchi (or Quimis) apparatus. Thin layer chromatography
was performed on silica gel plates (Merck 60F254). Infrared spectra
of 1% KBr pellets were recorded on a Bruker IFS66 spectrometer. 1H
NMR spectra were recorded on a Bruker AC 300 P spectrophotom-
eter in DMSO-d6 as the solvent, with tetramethylsilane as the inter-
nal standard. Mass spectra were recorded on an HCTultra Bruker
Daltonics spectrometer on the ESI positive ion polarity.
through a mechanism mediated by PPARc. This hypothesis was
also reinforced by the structural similarity between rosiglitazone
and the arylidene-thiazolidinediones evaluated in this study.
Finally, the comparison between the hydrophilic and hydropho-
bic interactions observed in the docking results for ligands 11 (best
activity), 12 (worst activity) and rosiglitazone revealed the inter-
molecular reasons for the superior docking score and anti-inflam-
matory activity of compound 11.
4.1.2. 3-(2-Chloro-6-fluoro-benzyl)-thiazolidine-2,4-dione; 4
C10H7ClFNO2S. Yield: 27%. Mp: 90 °C. TLC (n-hexane/ethyl ace-
tate, 9:1) Rf 0.4. IR (KBr, cmÀ1): 1755, 1685, 1600, 1341, 971,
784. 1H NMR 300 MHz (d ppm, DMSO-d6): 4.22 (s, 2H, CH2), 4.81
(s, 2H, NCH2), 7.19–7.26 (m, 1H, pos. 5 benzyl), 7.3–7.34 (m, 1H,
pos. 4 benzyl), 7.35–7.43 (m, 1H, pos. 3 benzyl). MS, ESI+: m/z
260 [M+H]+, 262 [M+H+2]+, 277 [M+NH4]+, 279 [M+NH4+2]+, 282
[M+Na]+, 284 [M+Na+2]+, 298 [M+K]+, 300 [M+K+2]+, 219, 143, 113.
4. Methods
4.1. Synthesis
Thiazolidine-2,4-dione (1) was N-(3)-alkylated in the presence
of sodium hydroxide, which allows the thiazolidine sodium salt
to react with the benzyl halide in a hot ethanol medium, yielding
intermediates 2, 3 and 4 as described in Scheme 1. The 5-arylid-
ene-3-benzyl-thiazolidine-2,4-diones were prepared by a nucleo-
philic Michael addition of the 3-benzyl-thiazolidine-2,4-dione 2,
3 or 4 and the respective aryl-substituted ethyl-(2-cyano-3-phe-
nyl)-acrylates20 to obtain the arylidene-thiazolidine-2,4-diones
5–12 (Scheme 1). After cooling, the precipitates were purified by
column chromatography or crystallized in suitable solvents. The
ethyl-(2-cyano-3-phenyl)-acrylates were prepared by Knoevenagel
condensation of ethyl cyanoacetic ester and substituted benzalde-
hydes in the presence of piperidine. The arylidene-thiazolidine-
2,4-diones were isolated in a single isomeric form, which was ver-
ified by TLC and NMR analysis. X-ray crystallographic studies and
13C NMR have demonstrated a preference for the Z configuration
for 5-arylidenethiazolidinones21–23 (Guarda et al., 2003; Tan
et al., 1986; Albuquerque et al., 1995).
4.1.3. Ethyl-3-(4-methanesulfonyl-phenyl)-2-cyanoacrylate
C13H13NO4S. Yield: 70%. Mp: 125–126 °C. TLC (benzene/ethyl
acetate, 1:1) Rf 0.76. IR (KBr, cmÀ1): 2222, 1728, 1607, 1301,
1264, 1197, 766. 1H NMR 300 MHz (d ppm, DMSO-d6): 1.32 (t,
3H, CH3 ester, J = 6.9 Hz), 4.35 (q, 2H, CH2 ester, J = 6.9 Hz), 3.3 (s,
3H, SO2CH3), 8.12 (d, 2H, pos. 2, 6 phenyl, J = 8.7 Hz), 8.23 (d, 2H,
pos. 3, 5 phenyl), 8.53 (s, 1H, ethylene).
4.1.4. Ethyl-3-(9H-fluoren-2-yl)-2-cyanoacrylate
C19H15NO2. Yield: 98%. Mp: 145–146 °C. TLC (n-hexane/ethyl
acetate, 7:3) Rf 0.65. IR (KBr, cmÀ1): 2217, 1717, 1598, 1268,
1241, 1214, 1119. 1H NMR 300 MHz (d ppm, DMSO-d6): 1.32 (t,
3H, CH3 ester, J = 7.2 Hz), 4.06 (s, 2H, CH2 fluorene), 4.33 (q, 2H,
CH2 ester, J = 6.9 Hz), 7.4–7.5 (m, 2H, pos. 6, 7 fluorenylidene),
7.64–7.69 (m, 1H, pos. 3 fluorenylidene), 8.03–8.07 (m, 1H, pos. 4
fluorenylidene), 8.1–8.17 (m, 2H, pos. 1, 8 fluorenylidene), 8.32–
8.35 (m, 1H, pos. 5 fluorenylidene), 8.47 (s, 1H, ethylene).
4.1.1. General procedures
4.1.5. 5-(5-Bromo-2-methoxy-benzylidene)-3-(3-chloro-benzyl)
-thiazolidine-2,4-dione; 5
3-Benzyl-thiazolidine-2,4-diones (2–4) general procedure: An
equimolar solution of sodium hydroxide in an ethanol/water mix-
ture (6:4) was added dropwise with stirring to a suspension of thi-
azolidine-2,4-dione in the same ethanol/water mixture. A few
minutes later, the substituted benzyl chloride was added and the
mixture was stirred for a few minutes before being refluxed for
24 h. After cooling at room temperature, the expected compound
was precipitated by addition of crushed ice before purification by
flash chromatography on silica with chloroform/methanol (92:8)
as the eluent. The published chemical data on 3-chloro-benzyl-thi-
C18H13BrClNO3S. Yield: 99%. Mp: 139–140 °C. TLC (n-hexane/
ethyl acetate, 7:3) Rf 0.68. IR (KBr, cmÀ1): 1336, 1382, 1479,
1598, 1691, 1744. 1H NMR 300 MHz (d ppm, DMSO-d6): 3.9 (s,
3H, OCH3), 4.83 (s, 2H, CH2), 7.24–7.29 (m, 1H, pos. 5 benzyl),
7.39 (1H, pos. 2 benzyl), 7.39 (dd, 2H, pos. 4, 6 benzyl, J = 4.5 and
1.2 Hz), 7.15 (d, 1H, pos. 3 benzylidene, J = 9.3 Hz), 7.55 (d, 1H,
pos. 6 benzylidene, J = 2.4 Hz), 7.67 (dd, 1H, pos. 4 benzylidene,
J = 8.7 and 2.4 Hz), 7.97 (s, 1H, ethylene). MS, ESI+: m/z 438
[M+H]+, 440 [M+H+2]+, 460 [M+Na]+, 462 [M+Na+2]+, 268, 122.
azolidine-2,4-dione24
2 and 2-bromo-benzyl-thiazolidine-2,4-
dione25 3 are not reported here.
4.1.6. 3-(3-Chloro-benzyl)-5-(4-methanesulfonyl-benzylidene)-
thiazolidine-2,4-dione; 6
C18H14ClNO4S2. Yield: 79%. Mp: 196–197 °C. TLC (n-hexane/
ethyl acetate 6:4) Rf 0.66. IR (KBr, cmÀ1): 1151, 1383, 1607, 1697,
1762. 1H NMR 300 MHz (d ppm, DMSO-d6): 3.28 (s, 3H, SO2CH3),
4.86 (s, 2H, CH2), 7.27–7.31 (m, 1H, pos. 5 benzyl), 7.38 (d, 1H,
Ethyl-(2-cyano-3-phenyl)-acrylates general procedure: An equi-
molar (23 mMol) mixture of aldehyde and ethyl cyanoacetate, in
the presence of piperidine (three drops) and benzene (20 mL),
was heated at 110–120 °C for 8–10 h. After cooling, the mixture
was caught in mass. The solid phase was recrystallized from an

3810
C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
pos. 2 benzyl, J = 1.5 Hz), 7.39–7.42 (m, 2H, pos. 4, 6 benzyl), 7.89
(d, 2H, pos. 2, 6 benzylidene, J = 8.7 Hz), 8.07 (d, 2H, pos. 3, 5 ben-
zylidene, J = 8.4 Hz), 8.06 (s, 1H, ethylene). MS, ESI+: m/z 408
[M+H]+, 410, 425 [M+NH4]+, 427 [M+H+2]+, 430 [M+Na]+, 432
[M+Na+2]+, 446 [M+K]+, 448 [M+K+2]+, 268, 154, 122.
J = 7.8 and 1.2 Hz), 7.91 (d, 2H, pos. 2, 6 benzylidene, J = 8.1 Hz),
8.09 (d, 2H, pos. 3, 5 benzylidene, J = 8.4 Hz), 8.08 (s, 1H ethylene).
MS, ESI+: m/z 452 [M+H]+, 409, 382, 296, 169.
4.1.12. 3-(2-Chloro-6-fluoro-benzyl)-5-(2,4-dichloro-
benzylidene)-thiazolidine-2,4-dione; 12
4.1.7. 5-Bipheny-4-ylmethylene-3-(3-chloro-benzyl)-thiazo-
lidine-2,4-dione; 7
C17H9Cl3FNO2S. Yield: 76%. Mp: 169–170 °C. TLC (n-hexane/
ethyl acetate, 7:3) Rf 0.8. IR (KBr, cmÀ1): 1335, 1377, 1601, 1689,
1742. 1H NMR MHz (d ppm, DMSO-d6): 4.99 (s, 2H, CH2), 7.23–
7.29 (m, 1H pos. 5 benzyl), 7.33–7.38 (m, 1H, pos. 4 benzyl),
7.39–7.47 (m, 1H, pos. 3 benzyl), 7.6 (d, 1H, pos. 5 benzylidene,
J = 8.7 Hz), 7.63 (d, 1H, pos. 6 benzylidene, J = 8.7 Hz), 7.86–7.88
(m, 1H, pos. 3 benzylidene), 7.95 (s, 1H, ethylene). MS, ESI+: m/z
416 [M+H]+, 418 [M+H+2]+, 438 [M+Na]+, 440 [M+Na+2]+, 302, 268.
C23H16ClNO2S. Yield: 73%. Mp: 169–170 °C. TLC (n-hexane/ethyl
acetate; 7:3) Rf 0.77. IR (KBr, cmÀ1): 1148, 1383, 1481, 1595, 1670,
1740. 1H NMR 300 MHz (d ppm, DMSO-d6): 4.86 (s, 2H, CH2), 7.27–
7.31 (m, 1H, pos. 5 benzyl), 7.39–7.42 (m, 2H, pos. 4, 6 benzyl), 7.39
(d, H, pos. 2 benzyl, J = 1.2 Hz), 7.44 (t, 1H, pos. 4 phenyl, J = 1.2 Hz),
7.51 (t, 2H, pos. 3 phenyl, J = 7.5 Hz), 7.77 (dd, 2H, pos. 2 phenyl,
J = 6.3 and 1.8 Hz), 7.74 (d, 2H, pos. 2 benzylidene, J = 8.4 Hz),
7.88 (d, 2H, pos. 3 benzylidene, J = 8.4 Hz), 8.03 (s, 1H, ethylene).
MS, ESI+: m/z 406 [M+H]+, 408 [M+H+2]+, 428 [M+Na]+, 430
[M+Na+2]+, 268, 122.
4.2. Biological tests
The anti-inflammatory effect was tested by the production of
air pouches on the dorsal cervical region of mice of 25–30 g by a
subcutaneous injection of 2.5 mL of sterile air on day 0, followed
by a second injection of 2.5 mL of sterile air 3 days later. On day
6, the 1 mL of 1% (w/v) carrageenan solution was injected into
the cavity. The arylidene-thiazolidinedione compounds (5–12)
and the reference drug rosiglitazone were administered orally 1 h
before the injection of carrageenan. After 6 h, the mice were sacri-
ficed by ether exposure, and the pouches were washed with 3 mL
4.1.8. 5-(3-Bromo-4-methoxy-benzylidene)-3-(3-chloro-benzyl)
-thiazolidine-2,4-dione; 8
C18H13BrClNO3S. Yield: 71%. Mp: 153–154 °C. TLC (n-hexane/
ethyl acetate, 6:4) Rf 0.83. IR (KBr, cmÀ1): 1268, 1327, 1376,
1495, 1587, 1684, 1742. 1H NMR 300 MHz (d ppm, DMSO-d6):
3.93 (s, 3H, OCH3), 4.84 (s, 2H, CH2), 7.24–7.28 (m, 1H, pos. 5 ben-
zyl), 7.38 (dd, 2H, pos. 4, 6 benzyl, J = 4.8 and 1.2 Hz), 7.39 (1H, hid-
den, pos. 2 benzyl), 7.30 (d, 1H, pos. 3 benzylidene, J = 9 Hz), 7.64
(dd, 1H, pos. 2 benzylidene, J = 9 and 2.4 Hz), 7.91 (d, 1H, pos. 2
benzylidene, J = 2.4 Hz), 7.93 (s, 1H, ethylene). MS, ESI+: m/z 438
[M+H]+, 440 [M+H+2]+, 460 [M+Na]+, 462 [M+Na+2]+, 309, 268,
122.
of saline solution containing 3 lM of EDTA. The number of mi-
grated neutrophils were determined by staining with Turk’s solu-
tion (0.01% crystal violet in 3% acetic acid) and counted using a
Neubauer hemocytometer.
4.3. Docking
4.1.9. 3-(3-Chloro-benzyl)-5-(9H-fluoren-2-yl-methylene)-
thiazolidine-2,4-dione; 9
The structures of the arylidene-thiazolidinediones 5–12 shown
in Scheme 1 were initially optimized using the AM1 method12
implemented with the BioMedCache program28 with the default
values for the convergence criteria. The preference for the Z config-
uration of the exocyclic double bond of the 5-arylidenethiazolidi-
nones was confirmed. Before the docking calculation, the
geometries of the arylidene-thiazolidinediones 5–12 were subse-
quently optimized using the Tripos force field available in the SYBYL
package.18
C24H16ClNO2S. Yield: 81%. Mp: 185–186 °C. TLC (n-hexane/ethyl
acetate, 7:3) Rf 0.74. IR (KBr, cmÀ1): 1331, 1380, 1595, 1683, 1733.
1H NMR 300 MHz (d ppm, DMSO-d6): 4.03 (s, 2H, CH2), 4.86 (s, 2H,
NCH2), 7.27–7.31 (m, 1H, pos. 5 benzyl), 7.39 (d, H, pos. 2 benzyl,
J = 1.2 Hz), 7.4 (dd, 2H, pos. 4, 6 benzyl, J = 7.5 and 1.8 Hz), 7.39–
7.45 (m, 2H, pos. 6, 7 fluorenylidene), 7.64 (d, 1H, pos. 3 fluoreny-
lidene, J = 6.9 Hz), 7.69 (d, 1H, pos. 5 fluorenylidene, J = 7.8 Hz),
7.86 (s, 1H, pos. 1 fluorenylidene), 7.99 (d, 1H, pos. 8 fluorenylid-
ene, J = 7.5 Hz), 8.08 (d, 1H, pos. 4 fluorenylidene, J = 8.1 Hz), 8.06
(s, 1H, ethylene). MS, ESI+: m/z 418[M+H]+, 420 [M+H+2]+, 440
[M+Na]+, 442 [M+Na+2]+, 304, 282, 168.
The potential affinities of those compounds with respect to the
PPAR
enzyme Peroxisome Proliferator-Activated Receptor Gamma
(PPAR ) co-crystallized with 5-[4-(2-[methyl(pyridin-2-yl)amino]
c structure were evaluated through docking studies using the
c
4.1.10. 3-(3-Chloro-benzyl)-5-(4-methoxy-benzylidene)-
thiazolidine-2,4-dione; 10
ethoxy)benzyl]thiazolidine-2,4-dione (rosiglitazone-BRL) as the
target. This complex was obtained from the RCSB Protein Data
Bank29 under the PDB code 2PRG. The FlexX 7.2 Program16 was used
for these computations because it takes into account the ligand flex-
ibility (main torsions) during the calculations.30 The structure of the
‘A’ monomer of the homodimer was chosen as the target for docking
studies. The sitewas definedas all atoms within a radiusof 6.5Å from
the co-crystallized rosiglitazone ligand. Additionally, the rosiglitaz-
one ligand was re-docked to test the program protocol. The theoret-
ical binding profile proposed for the thiazolidinedione ligands was
determined as the highest (most negative) score among 30 possible
solutions generated according to the FlexX16 scoring function and
protocol. Therefore, the docking results presented here are just the
highest score for each molecule studied.
C18H14ClNO3S. Yield: 72%. Mp: 125–126 °C. TLC (n-hexane/ethyl
acetate, 7:3) Rf 0.8. IR (KBr, cmÀ1): 1258, 1325, 1374, 1509, 1590,
1673, 1733. 1H NMR 300 MHz (d ppm, DMSO-d6): 3.84 (s, 3H,
OCH3), 4.84 (s, 2H, CH2), 7.24–7.3 (m, 1H, pos. 5 benzyl), 7.39 (d,
1H, pos. 2 benzyl, J = 1.8 Hz), 7.39 (dd, 2H, pos. 4, 6 benzyl, J = 5.1
and 1.2 Hz), 7.12 (d, 2H, pos. 3, 5 benzylidene, J = 8.7 Hz), 7.61 (d,
2H, pos. 2, 6 benzylidene, J = 8.7 Hz), 7.93 (s, 1H, ethylene). MS,
ESI+: m/z 360 [M+H]+, 362 [M+H+2]+, 382 [M+Na]+, 384
[M+Na+2]+, 332, 282.
4.1.11. 3-(2-Bromo-benzyl)-5-(4-methanesulfonyl-benzylidene)
-thiazolidine-2,4-dione; 11
C18H14BrNO4S2. Yield: 56%. Mp: 59–60 °C. TLC (n-hexane/ethyl
acetate, 1:1) Rf 0.69. IR (KBr, cmÀ1): 1149, 1306, 1383, 1603,
1678, 1748. 1H NMR 300 MHz (d ppm, DMSO-d6): 3.25 (s, 3H,
CH3), 4.87 (s, 2H, CH2), 7.24 (dd, 1H, pos. 6 benzyl, J = 7.8 and
1.2 Hz), 7.27 (dt, 1H, pos. 4 benzyl, J = 7.8 and 1.2 Hz), 7.36 (dt,
1H, pos. 5 benzyl, J = 7.5 and 1.2 Hz), 7.67 (dd, 1H, pos. 3 benzyl,
4.4. PPARc-competition binding assay
Polyhistidine-tagged human PPAR
(His-LBD-hPPAR ) was overexpressed in Escherichia coli BL21 cells
using the pET28a expression plasmid (the details of this construct
c ligand-binding domain
c

C. D. Barros et al. / Bioorg. Med. Chem. 18 (2010) 3805–3811
3811
have been described previously31) and purified by immobilized
metal ion affinity chromatography on a Co2+-loaded HiTrap chelat-
ing column (Clontech).
5. Petersen, K. F.; Krssak, M.; Inzucchi, S.; Cline, G. W.; Dufour, S.; Shulman, G. I.
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By performing saturating binding studies with purified His-
LBD-hPPAR
determined that the dissociation constant (Kd) of rosiglitazone
for PPAR was 33 nM, which is consistent with previously reported
c
and increasing amounts of [3H]rosiglitazone, we
c
values.32 For the competition binding assays, His-LBD-hPPAR
c was
11. Flower, R. J. Nat. Rev. 2003, 2, 179.
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incubated for 12 h at 4 °C with 40 nM [3H]rosiglitazone (specific
activity of 50 Ci/mmol) in buffer containing 10 mM Tris, pH 8.0,
50 mM KCl, and 10 mM dithiothreitol in a final volume of 100 lL.
13. Sakamoto, J.; Kimura, H.; Moriyama, S.; Odaka, H.; Momose, Y.; Sugiyama, Y.;
Sawada, H. Biophys. Res. Commun. 2000, 278, 704.
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Muscat, G. E. O.; Gustafson, T. A. Diabetes 2006, 55, 2523.
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www.tripos.com.
19. DeLano, W. L. The PyMOL Molecular Graphics System 2002, DeLano Scientific,
San Carlos, CA (USA), http://pymol.sourceforge.net.
20. Mourão, R. H.; Silva, T. G.; Soares, A. L. M.; Vieira, E. S.; Santos, J. N.; Lima, M. C.
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Vehicle (DMSO) or unlabeled ligand was then added. After incuba-
tion for an additional 12 h at 4 °C, bound radioactivity was sepa-
rated from free radioactivity by gravity flow through a 1 mL
Sephadex G-25 desalting column (Amersham) and quantitated
using a liquid scintillation counter (Perkin–Elmer). Concentra-
tion-dependent experiment results were expressed as the percent
[3H]rosiglitazone bound compared to the assay tube in which vehi-
cle was added. Binding curves were fit to a nonlinear regression,
and IC50 values were obtained using a one-site competition equa-
tion using the GraphPad 4.0 software (GraphPad, San Diego, CA).
Inhibition constant (Ki) values were calculated by the equation of
Cheng and Prussof.33
Acknowledgments
24. Muto, S.; Kubo, A.; Itai, A.; Sotome, T.; Yamaguchi, Y. PCT Int. Appl. 2005, WO
2005026127.
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We would like to thank the Brazilian National Research Council
(CNPq), INCT_if (Instituto Nacional em Ciência e Tecnologia para
Inovação Farmacêutica), and the Brazilian Foundation Coordenação
de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) for sup-
port for this joint research program.
28. BioMedCache version 6.1, CopyrightÓ 2000–2003 Fujitsu Limited, CopyrightÓ
1989–2000, Oxford Molecular Ltd, http://www.CACheSoftware.com.
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