Thiazolidin-4-ones from 4-(methylthio)benzaldehyde and 4-(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and?cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2016.08.057

The present study assessed the biological potential of fourteen 1,3-thiazolidin-4-ones evaluating the antiglioma effect through decreasing of cell viability of glioblastoma multiform cells. The new compounds were efficient synthesized through multicomponent or multicomponent one-pot procedures in moderate to good yields (22–86%) from two arenealdehydes (4-(methylthio)benzaldehyde and 4-(methylsulfonyl)benzaldehyde), seven amines (aromatic and aliphatic) and mercaptoacetic acid. The compounds were identified and characterized by GC/MS and NMR, five of them by HRMS. Six thiazolidinones showed significant effect of decreasing cell viability compared to standard drug TMZ at 100?μM in 72?h in C6 cell line by MTT assay. The compounds 5b, 5e, 5g and 6e showed the best results in the screening at 100?μM and were analyzed at different concentrations (5, 25, 50, 100 and 250?μM). Compounds 5b and 5e showed statistical difference at 5?μM, 6e at 25?μM and 5g at 50?μM in 72?h of treatment. The cytotoxicity study in primary astrocytes cells was evaluated and none of fourteen compounds showed toxicity at 100?μM, eight of them were not cytotoxic at 250?μM, both in 72?h. In addition, the propidium iodide assay demonstrated that the compounds might induce cell death by necrosis. In conclusion, this work reports at least four compounds (5b, 5e, 5g and 6e) with potential anti-tumor effect against glioblastoma multiform cell presenting activity at low concentrations and safe profile of cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2016.08.057

Accepted Manuscript
Thiazolidin-4-ones from 4-(methylthio)benzaldehyde and 4-
(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and cytotoxicity
Daniel Schuch da Silva, César Emiliano Hoffmann da Silva, Mayara Sandrielly
Pereira Soares, Juliana Hofstatter Azambuja, Taíse da Rosa Carvalho, Geórgia
Cristiane Zimmer, Clarissa Piccinin Frizzo, Elizandra Braganhol, Roselia Maria
Spanevello, Wilson Cunico
PII:
S0223-5234(16)30713-9
10.1016/j.ejmech.2016.08.057
EJMECH 8853
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2016
Revised Date: 22 August 2016
Accepted Date: 24 August 2016
Please cite this article as: D.S. da Silva, C.E.H. da Silva, M.S.P. Soares, J.H. Azambuja, T. da Rosa
Carvalho, G.C. Zimmer, C.P. Frizzo, E. Braganhol, R.M. Spanevello, W. Cunico, Thiazolidin-4-ones from
4-(methylthio)benzaldehyde and 4-(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and
cytotoxicity, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.057.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Thiazolidin-4-ones from 4-(methylthio)benzaldehyde and 4-
(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and
cytotoxicity
O
Efficient synthesis with regular to good yields (22 - 86%)
N
S
R1
In vitro anti-glioma C6 assay
best active 5b, 5e, 5g and 6e
safe therapeutic profile
Cytotoxicity assay in primary astrocytes
Propidium iodide assay suggest cell death mediated by necrosis
R2
N
R1
R2
O
O
S
S
O
N
O
5
6
g
e
b

ACCEPTED MANUSCRIPT
Thiazolidin-4-ones
from
4-(methylthio)benzaldehyde
and
4-
(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and cytotoxicity
Daniel Schuch da Silvaa,b, César Emiliano Hoffmann da Silvaa,b, Mayara Sandrielly
Pereira Soaresa,c, Juliana Hofstatter Azambujaa,c, Taíse da Rosa Carvalhoa,c, Geórgia
Cristiane Zimmerd, Clarissa Piccinin Frizzod, Elizandra Braganhola,c, Roselia Maria
Spanevelloa,c, Wilson Cunico*a,b
a
Programa de Pós-Graduação em Bioquímica e Bioprospecção, Centro de
Ciências Químicas Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, RS,
Brasil.
b
Laboratório de Química Aplicada a Bioativos, Centro Ciências Químicas,
Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, RS, Brasil.
c Laboratório de Neuroquímica, Inflamação e Câncer, Centro de Ciências Químicas
Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, RS, Brasil.
d
Núcleo de Estudos Químicos e Complexidade Molecular (NEOQCOM),
Universidade Federal de Santa Maria, RS, Brasil.
* Corresponding author
E-mail address: wjcunico@yahoo.com.br
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Abstract
The present study assessed the biological potential of fourteen 1,3-thiazolidin-4-
ones evaluating the antiglioma effect through decreasing of cell viability of glioblastoma
multiform cells. The new compounds were efficient synthesized through multicomponent or
multicomponent one-pot procedures in moderate to good yields (22-86%) from two
arenealdehydes (4-(methylthio)benzaldehyde and 4-(methylsulfonyl)benzaldehyde), seven
amines (aromatic and aliphatic) and mercaptoacetic acid. The compounds were identified
and characterized by GC/MS and NMR, five of them by HRMS. Six thiazolidinones
showed significant effect of decreasing cell viability compared to standard drug TMZ at
100 µM in 72h in C6 cell line by MTT assay. The compounds 5b, 5e, 5g and 6e showed
the best results in the screening at 100 µM and were analyzed at different concentrations
(5, 25, 50, 100 and 250 µM). Compounds 5b and 5e showed statistical difference at 5 µM,
6e at 25 µM and 5g at 50 µM in 72h of treatment. The cytotoxicity study in primary
astrocytes cells was evaluated and none of fourteen compounds showed toxicity at 100
µM, eight of them were not cytotoxic at 250 µM, both in 72h. In addition, the propidium
iodide assay demonstrated that the compounds might induce cell death by necrosis. In
conclusion, this work reports at least four compounds (5b, 5e, 5g and 6e) with potential
anti-tumor effect against glioblastoma multiform cell presenting activity at low
concentrations and safe profile of cytotoxicity.
Keywords: thiazolidin-4-ones, glioblastoma multiform, astrocytes, anti-tumor activity,
necrosis.
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1 Introduction
Central nervous system (CNS) tumors are the second leading cause of death from
neurological disease [1]. Among them, gliomas are the most common primary brain tumors
in adults, standing out glioblastoma multiform (GBM) as the more prevalent and the worst
prognosis among malignant brain tumors. GBMs are grade IV gliomas and are
chemoresistance to currently used therapy and because of its pattern of infiltrative growth
and high rate of proliferation, the effective treatment becomes extremely difficult [1-3].
Treatment of gliomas involves surgical removal, radiation and chemotherapy with
current used therapy temozolomide (TMZ), an alkylating agent that induces DNA damage
leading to cell death [4,5]. For tumor treatment, radiotherapy is the most important,
increasing patient survival of 3 to 4 months for 7 to 12 months. Still, the use of adjuvant
TMZ increases the survival from an average of 12.1 to 14.6 months [6].
However, it has been important GBM resistance from TMZ, promoted by various
DNA repair pathways. In this case, there is currently no other treatments for resistant
GBM, leading to an extremely poor prognosis for the patient [7]. Considering these
resistances of current therapy, it is important the development of new bioactive
compounds. Five-membered heterocycles containing nitrogen and sulfur, e.g.
thiazolidinones, have been extremely investigated to possess important chemical
properties and a great versatility of biological activities, like anti-inflammatory [8,9],
antimicrobial [10], antifungal [11,12], anticonvulsant [13], antioxidant [14], among others
[15,16]. A large number of compounds containing this heterocyclic moiety have also been
reported in the literature for possess potential anti-tumor activity [17-21], however, to the
best of our knowledge, it is uncommon the study of thiazolidinones against glioma cells.
Authors have been reported anti-tumor activity of compounds linking thiazolidinone
with other heterocycle. Kamel et al [17] reported 1,3-thiazolidin-4-ones containing indole
and pyridine groups with better IC50 than Doxorubicin against HeLa and MCF7 cell lines.
Furthermore, Wang et al [18] synthesized 1,3-thiazolidin-4-ones with indolin-2-one moiety
from 3-(diethylamino)propylamine and 3,4-(methylenedioxy)benzaldehyde and some
compounds showed better IC50 values than Sunitinib in four different human cancer cell
lines.
Given the above, this work aims to study new compounds that could be act as a
prominent therapeutic targets for anti-tumor activity. Therefore, continuing the study of our
research group regarding thiazolidinones, the aims of this study are: the synthesis of such
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heterocycles from seven different amines, two arenaldehydes and mercaptoacetic acid;
the evaluation of anti-tumor effect in GBM cells (C6); and the cytotoxicity in primary
astrocytes.
2 Results and Discussion
Fourteen new 1,3-thiazolidin-4-ones were synthesized from multicomponent (5a-d,
6a-d, 5f-g and 6f-g) or multicomponent one-pot (5e and 6e) reactions between primary
amines 3a-g, two aldehydes 1 or 2 and mercaptoacetic acid 4 (Scheme 1). All reactions
were realized through conventional thermal heating methodology, in toluene refluxing
using a Dean-Stark apparatus for water removal by azeotropic distillation. All compounds
were obtained in regular to good yields and have been properly characterized.
In general, the yields obtained after purification varied from regular to good and the
procedures were considerate efficient, in view of the obtaining of all proposed products. All
compounds were identified and characterized by mass spectrometry (GC-MS) and nuclear
magnetic resonance (NMR) of 1H and 13C. Besides that, compounds 5b, 5c, 5e, 5f and 6c
were also characterized by high resolution mass spectrometry (HRMS). It is emphasized
that all analyses are in full agreement with the proposed structures.
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Scheme 1. Reagents and synthetic conditions of proposal thiazolidinones
It is important to note the difference in lipophilicity of thiazolidinones, where the
compounds from 4-(methylthio)benzaldehyde 5 posses the value of Log P clearly higher
than compounds from 4-(methylsulfonyl)benzaldehyde 6 (see experimental 4.1.2). The
lipophilicity may affect the oral absorption and the penetration of the blood brain barrier.
This physico-chemical parameter is important once glioblastoma is a CNS tumor. The
lipophilicity could be adjusted by pharmacological or biotechnological techniques, including
nanocapsules.
After, the anti-tumor assay of all synthesized thiazolidinones was carried out in
glioblastom C6 cell line. Firstly, the results of the screening at 100 µM in 48h and 72h of
exposure are shown in Figure 1, as percentage of cell viability considering the mean of
DMSO control group as 100%. The compound 5g (61.2%) was the only that show
statistical difference from the positive control TMZ (42.3%), however compounds 5b
(52.2%), 5e (48.0%) and 6e (47.2%) also showed good results of inhibition of tumor cell
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viability at 100 µM in 48h. In 72h, all compounds demonstrated difference from DMSO at
100 µM and it is important to note that 5a (61.0%), 5b (78.0%), 5c (67.5%), 5e (75.0%), 5g
(81.7%) and 6e (77.0%) showed statistical difference in decreasing of cell viability when
compared to TMZ (39.7%).
MTT as C6 cell line treatment- 48h
150
100
*
*
**
***
***
***
***
#
***
50
***
0
MTT as C6 cell line treatment- 72h
150
#
100
50
0
#
*
#
#
*
*
**
**
***
***
***
***
#
#
***
***
#
#
***
#
***
#
***
***
Figure1. Anti-tumor screening of thiazolidinones (100 µM) in 48h and 72h of exposure.
Bars represent means ± SEM; * represents statistical difference from DMSO control group,
considering P<0.05; **P<0.01; ***P<0.001; # represents statistical difference from TMZ
(100 µM) positive control group, considering P<0.05 (One-way ANOVA followed by post-
hoc Tukey’s test, n=4).
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Considering the results of Figure 1, the four best active thiazolidinones 5b, 5e, 5g
and 6e were carried out to a concentration-responding test, as shown in Figure 2, and it
can be observed the importance of dose for the activity. In 48h, compounds 5b and 5e
showed inhibition since 100 µM, 5g since 50 µM and 6e since 25 µM. In 72h, compounds
5g and 6e maintained its activity since 50 µM and 25 µM, respectively. The best results
were found for 5b and 5e that presented significant difference at 5 µM, the lowest
concentration tested. So, it is clear that the time was important for the activity of 5b and 5e
(compare 48h to 72h). This fact could be explained by the longer contact time of the
compounds with the GBM cells. These results reinforce the potential of 1,3-thiazolidin-4-
ones as anti-tumor agents, already reviewed by Jain et al in 2012 [16] and Tripathi et al in
2014 [15].
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A
Concentration-response effect of 5b - 48H
0.4
Concentration-response effect of 5e - 48h
0.4
0.3
0.3
***
***
0.2
0.2
***
0.1
0.1
***
0.0
0.0
Concentration-response effect of 5g - 48h
0.4
Concentration-response effect of 6e - 48h
0.4
0.3
0.3
***
***
***
0.2
0.2
0.1
0.0
***
***
***
***
0.1
0.0
B
Concentration-response effect of 5e - 72h
0.8
Concentration-response effect of 5b - 72h
0.8
0.6
0.6
**
***
***
0.4
0.4
***
***
***
0.2
0.2
***
***
***
***
0.0
0.0
Concentration-response effect of 6e - 72h
0.8
Concentration-response effect of 5g - 72h
0.8
0.6
0.6
*
***
***
0.4
0.4
***
***
0.2
0.2
***
***
0.0
0.0
Figure 2. Evaluation of inhibitory effect in different concentrations of thiazolidinones
5b, 5e, 5g and 6e in cell viability. A. C6 cells treatment with different concentrations of 5b,
5e, 5g and 6e in 48h. B. C6 cells treatment with different concentrations of 5b, 5e, 5g and
6e in 72h. Bars represent means ± SEM; *represents statistical difference from DMSO
group, P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA followed by Tukey’s test, n=4).
In general, it is clear the importance of 2-(4-methylthio)phenyl once compounds
bearing this group (5) showed better results than compounds 6 with 2-(4-
methylsunfonyl)phenyl group. From seven different amines studied, four of the best
compounds have the propylpiperidine (b), benzo-3,4-metilenedioxi (g) or methylpyridine
8

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(e) moiety. It can be noted that the substituent 3-methylpyridin-2-yl (e) could be important
for anti-tumor action, in view of the two compounds (5e and 6e) that possess this
substituent are among the top four. Furthermore, the compound 6e was clearly the best
compound of series 6, again demonstrating the importance of 3-methylpyridin-2-yl (e)
group to the activity.
It emphasized that there was reported in the literature thiazolidinone with pyridine
core with good anti-tumor effect [17], the same core contained in compounds 5e and 6e.
Other authors also reported thiazolidinones from 3-(diethylamino)propylamine with good
anti-tumor activity [18], similar to 1-(2-aminoethyl)piperidine (b) used for the synthesis of
compound 5b. This work also report thiazolidinones containing the benzo-[3,4]-
methylenedioxy core with good activity [18], a substituent core of compound 5g.
In addition, it was evaluated the cytotoxic effect of all compounds through the MTT
assay in primary astrocyte cells to verify the therapeutic window. The Figure 3 shows that
ten of fourteen compounds do not present statistical differences from DMSO in 72h at 250
µM. However, compounds 5a, 5b, 5c and 6c were cytotoxic at this concentration. It
emphasized that none compound present any toxicity at 100 µM, demonstrating clearly
safe profile at this concentration. Three (5e, 5g and 6e) of the top four in anti-tumor test
were not cytotoxic to normal cell in the highest concentration tested (250 µM) and only 5b
was considerate toxic at 250 µM.
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Primary astrocytes treatment 100 µM - 72H
200
150
100
50
***
***
**
0
Primary astrocytes treatment 250 µM - 72H
200
150
100
50
*
*
*
*
0
Figure 3. Evaluation of treatment of astrocytes cells with proposal thiazolidinones at 100
µM and 250 µM in 72h. *represents statistical difference from DMSO control group,
considering P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA followed by Tukey’s test,
n=3). Bars represent means ± SEM.
Following determination of the effective concentrations for antiglioma activity, it was
investigated a possibly pathway of cell death involved in the mechanism of action of active
compounds. The concentrations of 50 and 100 µM were chosen for these analyzes, once
significant effects were verified in decreasing of cell viability selectively in glioma lineage.
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The permeability of the cell membrane was assessed by propidium iodide
incorporation technique, marker of death by necrosis. All compounds were effective in
lowering the density and changing the cell morphology compared to DMSO control group
in both concentrations. Furthermore, incorporation of PI was observed, and the results
suggest the effects of the compounds are mediated by necrosis (Figure 4), however other
mechanism of cell death studies are needed to confirm this hypothesis.
Figure 4. Propidium iodide assay of C6 treatment for 72h with thiazolidinones 5b, 5e, 5g
and 6e in 50 and 100 µM. Fluorescence (right panel) and phase contrast (left panel)
microphotographs were taken in 20x magnification.
3 Conclusions
It is important to highlight the obtaining of fourteen new thiazolidinones that are fully
identified and characterized. All reaction conditions were efficient for the synthesis of the
desired compounds. Considering the poor prognosis and chemoresistance to therapy of
GBM, it makes extremely relevant to search for new therapeutic agents, e.g.
thiazolidinones. The anti-tumor evaluation results revealed at least four potential
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compounds (5b, 5e, 5g and 6e) with significant inhibition in low doses and good
therapeutic window, excellent agents for glioma treatment.
Lastly, studies are needed to confirm the pathways of cell death. The structure-
activity relationship shows that the both thiazolidinone and pyridine rings (5e and 6e) were
important to activity. Then it is emphasized that new studies have been grounded,
including in vivo trials to confirm the potential of these thiazolidinones as safe therapeutic
agents with anti-tumor effect to glioblastoma multiform cells.
4 Experimental
4.1 Chemistry
4.1.1 General
All common reagents and solvents were used as obtained from commercial
suppliers without further purification. Reactions progress was monitored by a thin- layer
chromatography (TLC) (hexane:ethyl acetate 3:1) and/or by a Shimadzu Gas
Chromatograph GC-2010, HP-1 column (cross linked methyl siloxane, 30 m × 0.32 mm ×
0.25 ꢀm): Column head pressure, 14 psi, program: T0 = 60 °C; t0 = 2.0 min; rate 10.0 °C
min–1; Tf = 280 °C; tf = 13.0 min; Inj. = 250 °C; D et. = 280 °C. The melting points were
determined using open capillaries on a Fisatom model 430 apparatus and are uncorrected.
1H and 13C NMR spectra were recorded on a Bruker 250 spectrometer (1H at 250 MHz and
13C at 62.5 MHz),on a Bruker DRX 400 spectrometer (1H at 400 MHz and 13C at 100 MHz),
13
on a Bruker Avance 500 spectrometer (1H at 500 MHz and C at 125 MHz), on a Bruker
13
Avance 600 spectrometer (1H at 600 MHz and C at 150 MHz), and on a Bruker Avance
III 600 MHz (1H at 600 MHz and 13C at 150 MHz), in CDCl3 containing TMS as an internal
standard or DMSO. The mass spectra were obtained on a Shimadzu GCMS-QP2010SE
with a split-splitless injector and equipped with a RDX-SMS capillary column (30 m x 0.25
mm x 0.25 ꢀm); helium was used as the carrier gas (56 kPa). The Log P values, or a
measure of lipophilicity, was calculated in ChemBioDraw Ultra 13.0 software. Mass
spectra were acquired in a GCT-Premier Waters (EI) equipment.
4.1.2 General procedure for the synthesis of thiazolidinones 5a-g and 6a-g
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The compounds 5a-c and 6a-c were obtained according Kunzler et al [12], 5d and
6d according Gouvêa et al [22], 5e and 6e according Neves et al [23] and 5g and 6g
according Masteloto et al [24].
To obtain the compounds 5f and 6f, the reaction proceeds by a mixture of 4-
(methylthio)aniline 3f (1mmol), arenaldehyde 1 or 2 (1 mmol) and PTSA (5% w/v) in
refluxing toluene for 3 hours using a Dean-Stark apparatus. After, was added the
mercaptoacetic acid 4 (3 mmol) and the reaction was heated for more 19 hours. For
compound 5f, the organic layer was neutralized with saturated NaHCO3 (3 x 30 ml), dried
with MgSO4 and concentrated to obtain the product. For compound 6f, the mixture was
concentrated and the solid compound was filtered off. Both products were purified by
column chromatography using hexane:ethyl acetate (8:2) as eluent.
The atom-numbering of synthesized compounds for NMR analysis identification are
given in Figure 5.
Figure 5. The atom-numbering for synthesized compounds
4.1.2.1 2-(4-(methylthio)phenyl)-3-(2-(piperidin-1-yl)ethyl)thiazolidin-4-one5a
1
Yield: 86,6%; Log P: 3.0; M.p,: 64-66 ºC; H NMR δ (CDCl3, 600MHz, ppm, J
=
H-H
3
3
Hz); 7.17 (d, 2H, J=8.5, Ar); 7.14 (d, 2H, J=8.5, Ar); 5.80 (d, 1H, 4J=1.5, H-2); 3.70 (dd,
2
4
2
1H, J=15.1, J=1.9, H-5a); 3.67-3.72 (m, 1H, H-6a); 3.63 (d, 1H, J=15.4, H-5b); 2.70
2
3
(ddd, 1H, J=13.9, J=6.9, H-6b); 2.40-2.45 (m, 1H, H-7a); 2.42 (s, 3H, -SCH3); 2.23 (br,
4H, pip.); 2.20 (ddd, 1H, 2J=12.4, 3J=6.1, H-7b); 1.47 (quint, 4H, 3J=5.6, pip.); 1.34 (br, 2H,
pip.).13C NMR δ (CDCl3, 150MHz, ppm, J C-F = Hz); 171.1 (C-4); 139.8 (Ar); 136.3 (Ar);
127.5 (2C, Ar); 126.6 (2C, Ar); 63.7 (C-2); 56.3 (C-7); 54.5 (2C, pip.); 39.8 (C-6); 32.9 (C-
5); 26.0 (2C, pip.); 24.2 (pip.); 15.5 (-SCH3). MS (70 eV): m/z (%) = 366 (M+, 1.7); 252
(0.5); 167 (2); 111 (3); 98 (100); 84 (4).
4.1.2.2 2-(4-(methylthio)phenyl)-3-(3-(piperidin-1-yl)propyl)thiazolidin-4-one5b
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Yield: 64,7%; Log P: 3.11; M.p.: 49-51 ºC; 1H NMR δ (CDCl3, 500MHz, ppm, J H-H
=
2
2
Hz); 7.16 (s, 4H, Ar); 5.60 (s, 1H, H-2); 3.72 (d, 1H, J=15.5, H-5a); 3.61 (d, 1H, J=15.3,
2
3
2
3
H-5b);3.57 (ddd, 1H, J=13.7, J=6.8, H-6a); 2.64 (ddd, 1H, J=13.9, J=6.9, H-6b); 2.41
(s, 3H, -SCH3); 2.19-2.24 (m, 5H, H-7a, pip.); 2.11-2.17 (m, 1H, H-7b); 1.59-1.67 (m, 1H,
H-8a); 1.52-1.57 (m, 1H, H-8b); 1.48 (quint, 4H, 3J=5.4, pip.); 1.35 (br, 2H, pip.). 13C NMR
δ (CDCl3, 125MHz, ppm, J C-F = Hz); 171.0 (C-4); 140.0 (Ar); 135.9 (Ar); 127.5 (2C, Ar);
126.5 (2C, Ar); 63.2 (C-2); 56.0 (C-7); 54.3 (2C, pip.); 41.2 (C-6); 32.9 (C-4); 25.7 (2C,
pip.); 24.2; 23.9; 15.4 (-SCH3). MS (70 eV): m/z (%) = 350 (M+, 3); 266 (0.6); 127 (5); 112
(3.5); 98 (100); 84 (9).C18H26N2OS2 (M+H)+: calculated 351.1565; found 351.1560.
4.1.2.3 3-(3-(diethylamino)propyl)-2-(4-(methylthio)phenyl)thiazolidin-4-one5c
1
Yield: 29,1%; Log P: 3.05; Oil; H NMR δ (CDCl3, 500MHz, ppm, J H-H = Hz); 7.16
(s, 4H, Ar); 5.58 (d, 1H,4J=1.4, H-2); 3.72 (d, 1H, 2J=15.5, H-5a); 3.62 (d, 1H, 2J=15.5, H-
2
3
2
3
3
5b);3.55 (ddd, 1H, J=14.4, J=7.3, H-6a); 2.66 (ddd, 1H, J=13.9, J=8.4, J=5.5, H-6b);
3
2.42 (q, 4H, J=7.1, -NEt); 2.41 (s, 3H, -SCH3); 2.26-2.36 (m, 2H, H-7); 1.56-1.64 (m, 1H,
H-8a); 1.46-1.55 (m, 1H, H-8b); 0.91 (t, 6H,3J=7.3, -NEt). 13C NMR δ (CDCl3, 100MHz,
ppm, J C-F = Hz); 171.0 (C-4); 140.0 (Ar); 135.9 (Ar); 127.5 (2C, Ar); 126.5 (2C, Ar); 63.2
(C-2); 48.9 (C-7); 46.4 (2C, -NEt); 41.2 (C-6); 32.9 (C-5); 24.2 (C-8); 15.4 (-SCH3); 11.1
(2C, -NEt). MS (70 eV): m/z (%) =338 (M+, 3); 309 (1.5); 266 (2.5); 115 (3.5); 98 (6); 86
(100); 72 (10).C17H26N2OS2 (M+H)+: calculated 339.1565; found 339.1549.
4.1.2.4 2-(4-(methylthio)phenyl)-3-(pyridin-2-yl)thiazolidin-4-one5d
Yield: 35,3%; Log. P: 3.31; M.p.: 109-111 ºC; 1H NMR δ (CDCl3, 500MHz, ppm, J H-
3
4
3
H = Hz); 8.16 (dd, 1H, J=4.8, J=1.1, pyridin); 7.96 (d, 1H, J=8.4, pyridin); 7.60 (td, 1H,
4
3
3J=7.9, J=1.9, pyridin); 7.14 (d, 2H, J=8.4, Ar); 7.05 (d, 2H,3J=8.4, Ar); 6.92 (ddd, 1H,
3J=7.3, 3J=4.9, 4J=0.7, pyridin);6.76 (s, 1H, H-2); 3.93 (dd, 1H, 2J=16.0, 4J=1.1, H-5a); 3.73
(d, 1H, 2J=16.0, H-5b); 2.34 (s, 3H, -SCH3). 13C NMR δ (CDCl3, 125MHz, ppm, J C-F = Hz);
171.2 (C-4); 150.6; 147.7; 138.5; 137.7; 137.6; 126.6 (2C, Ar); 126.3 (2C, Ar); 120.7;
117.3; 62.6 (C-2); 34.1 (C-5); 15.4 (-SCH3). MS (70 eV): m/z (%) =302 (M+, 46); 260 (30);
227 (50); 152 (60); 125 (55); 79 (100).
4.1.2.5 2-(4-(methylthio)phenyl)-3-(pyridin-2-ylmethyl)thiazolidin-4-one5e
14

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Yield: 62,6%; Log. P: 3.09; M.p.: 75-77 ºC; 1H NMR δ (CDCl3, 600MHz, ppm, J H-H
=
Hz); 8.45 (ddd, 1H, 3J=4.7, 5J=0.8, pyridin); 7.55 (td, 1H, 3J=7.6, 4J=1.8, pyridin); 7.08-7.15
4
(m, 6H, Ar, pyridin); 5.63 (d, 1H, J=1.7, H-2); 4.97 (d, 1H,2J=15.4, H-6a); 3.80 (dd, 1H,
4
2
2
2J=15.3, J=1.9, H-5a);3.78 (d, 1H, J=15.3, H-6b); 3.72 (d, 1H, J=15.6, H-5b); 2.41 (s,
3H, -SCH3). 13C NMR δ (CDCl3, 150MHz, ppm, J
= Hz); 171.4 (C-4); 155.4; 149.5;
C-F
140.0; 136.7; 135.4; 127.8 (2C, Ar); 126.6 (2C, Ar); 122.5; 122.3; 62.1 (C-2); 47.7 (C-6);
32.8 (C-5); 15.4 (-SCH3). MS (70 eV): m/z (%) =316 (M+, 0.5); 224 (7.5); 167 (4); 152 (9);
93 (100); 79 (3).C16H16N2OS2 (M+H)+: calculated 317.0782; found 317.0765.
4.1.2.6. 2,3-bis(4-(methylthio)phenyl)thiazolidin-4-one 5f
Yield: 45.6%; Log. P: 4.37; M.p.: 102-105 ºC; 1H NMR δ (CDCl3, 600MHz, ppm, J H-
H = Hz); 7.19 (d, 2H, 3J=7.7, Ar); 7.14 (d, 4H, 3J=7.1, Ar); 7.06 (d, 2H, 3J=8.0, Ar); 6.01 (s,
2
2
1H, H-2); 3.96 (d, 1H, J=15.9, H-5a); 3.86 (d, 1H, J=15.9, H-5b); 2.44 (s, 3H, -SCH3);
2.41 (s, 3H, -SCH3). 13C NMR δ (CDCl3, 150MHz, ppm, J C-F = Hz); 171.0 (C-4); 139.8 (Ar);
137.7 (Ar); 135.7 (Ar); 134.3 (Ar); 127.4 (2C, Ar); 126.9 (2C, Ar); 126.3 (2C, Ar); 126.1
(2C, Ar); 65.2 (C-2); 33.4 (C-5); 15.6 (-SCH3); 15.3 (-SCH3). MS (70 eV): m/z (%) = 347
(M+, 65); 273 (12); 225 (1.5); 178 (100); 169 (30); 135 (85).C17H17NOS3 (M+H)+: calculated
348.0536; found 348.0551.
4.1.2.7 3-(benzo[d][1,3]dioxol-5-yl)-2-(4-(methylthio)phenyl)thiazolidin-4-one 5g
Yield: 59,7%; Log. P: 3.71; M.p.: 98-100 ºC; 1H NMR δ (DMSO, 600MHz, ppm, J H-H
3
3
4
= Hz); 7.32 (d, 2H, J=8.4, Ar); 7.15 (d, 2H, J=8.3, Ar); 6.90 (d, 1H, J=2.0, Ar); 6.81 (d,
3
4
1H, J=8.3, Ar); 6.71 (dd, 1H,3J=8.3, J=2.0, Ar); 6.34 (s, 1H, H-2);5.97 (dd, 2H, J=8.7,
2
4
2
J=0.8, -OCH2O-); 3.97 (dd, 1H, J=15.7, J=1.5, H-5a); 3.85 (d, 1H, J=15.6, H-5b); 2.42
13
(s, 3H, -SCH3). C NMR δ (DMSO, 150MHz, ppm, J C-F = Hz); 170.3 (C-4); 147.1 (Ar);
145.5 (Ar); 138.6 (Ar); 136.1 (Ar); 131.3 (Ar); 127.8 (2C, Ar); 125.4 (2C, Ar); 119.5 (Ar);
107.7 (Ar); 107.3 (Ar); 101.4 (-OCH2O-); 63.3 (C-2); 32.5 (C-5); 14.1 (-SCH3). MS (70 eV):
m/z (%) =345 (M+, 60); 271 (20); 178 (75); 167 (50); 135 (100); 121 (13).
4.1.2.8 2-(4-(methylsulfonyl)phenyl)-3-(2-(piperidin-1-yl)ethyl)thiazolidin-4-one6a
1
Yield: 40,2%; Log P: 1.27; Oil; H NMR δ (CDCl3, 600MHz, ppm, J H-H = Hz); 7.96
3
3
2
(d, 2H, J=8.3, Ar); 7.51 (d, 2H, J=8.2, Ar); 6.03 (s, 1H, H-2); 3.92 (ddd, 1H, J=13.9,
2
4
2
3J=6.6, H-6a); 3.80 (dd, 1H, J=15.5, J=1.4, H-5a); 3.75 (d, 1H, J=15.5, H-5b); 3.06 (s,
15

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2
3
2
3
3H, -SO2CH3); 2.80 (ddd, 1H, J=13.6, J=6.6, H-6b); 2.64 (ddd, 1H, J=12.8, J=6.4, H-
7a); 2.55 (ddd, 1H, 2J=12.8, 3J=6.4, H-7b); 2.50 (br, 4H, pip.); 1.62 (quint, 4H, 3J=5.3, pip.);
1.46 (br, 2H, pip.). 13C NMR δ (CDCl3, 150MHz, ppm, J C-F = Hz); 171.5 (C-4); 146.2 (Ar);
141.0 (Ar); 128.2 (2C, Ar); 127.8 (2C, Ar); 62.6 (C-2); 55.2 (C-7); 54.1 (2C, pip.); 44.3 (-
SO2CH3); 38.3 (C-6); 32.6 (C-5); 24.9 (2C, pip.); 23.5 (pip.). MS (70 eV): m/z (%) =368
(M+, 0.7); 199 (0.5) 111 (2); 98 (100); 84 (2).
4.1.2.9 2-(4-(methylsulfonyl)phenyl)-3-(3-(piperidin-1-yl)propyl)thiazolidin-4-one6b
Yield: 35,4%; Log P: 1.37; M.p.: 71-73 ºC; 1H NMR δ (CDCl3), 600MHz, ppm, J H-H
=
3
3
Hz); 7.97 (d, 2H, J=8.3, Ar); 7.52 (d, 2H, J=8.3, Ar); 5.84 (d, 1H, 4J=1.2, H-2); 3.82 (dd,
2
4
2
1H, J=15.6, J=1.5, H-5a); 3.71-3.76 (m, 1H, H-6a); 3.72 (d, 1H, J=15.6, H-5b);3.09 (s,
2
3
3H, -SO2CH3); 2.69 (ddd, 1H, J=13.8, J=6.9, H-6b); 2.33-2.38 (m, 5H, H-7a, pip.); 2.24-
2.29 (m, 1H, H-7b); 1.73-1.79 (m, 1H, H-8a); 1.66-1.72 (m, 1H, H-8b); 1.58 (quint, 4H,
13
3J=5.6, pip.); 1.44 (br, 2H, pip.). C NMR δ (CDCl3, 125MHz, ppm, J C-F = Hz); 171.2 (C-
4); 146.2 (Ar); 141.0 (Ar); 128.2 (2C, Ar); 127.7 (2C, Ar); 62.4 (C-2); 55.6 (C-7); 54.2 (2C,
pip.); 44.3 (-SO2CH3); 41.3 (C-6); 32.6 (C-5); 25.5 (2C, pip.); 24.0; 23.7. MS (70 eV): m/z
(%) = 382 (M+, 2); 298 (0.5); 182 (2); 112 (3.5); 98 (100); 84 (6.5).
4.1.2.10 3-(3-(diethylamino)propyl)-2-(4-(methylsulfonyl)phenyl)thiazolidin-4-one6c
1
Yield: 22,3%; Log P: 1.32; Oil; H NMR δ (CDCl3, 500MHz, ppm, J H-H = Hz); 7.90
3
3
(d, 2H, J=8.2, Ar); 7.43 (d, 2H, J=8.3, Ar); 5.73 (d, 1H,4J=1.1, H-2); 3.75 (dd, 1H,
2J=15.5, 4J=1.6, H-5a); 3.63-3.69 (m, 1H, H-6a); 3.65 (d, 1H, 2J=15.5, H-5b);3.01 (s, 3H, -
2
3
3
3
SO2CH3); 2.63 (ddd, 1H, J=13.9, J=8.1, J=5.8, H-6b); 2.46 (q, 4H, J=7.1, -NEt); 2.31-
2.40 (m, 2H, H-7); 1.62-1.70 (m, 1H, H-8a); 1.53-1.61 (m, 1H, H-8b); 0.92 (t, 6H, 3J=7.1, -
13
NEt). C NMR δ (CDCl3, 100MHz, ppm, J C-F = Hz); 171.2 (C-4); 146.2 (Ar); 141.1 (Ar);
128.3 (2C, Ar); 127.7 (2C, Ar); 62.4 (C-2); 49.8 (C-7); 46.3 (2C, -NEt); 44.4 (-SO2CH3);
41.4 (C-6); 32.7 (C-5); 24.2 (C-8); 10.9 (2C, -NEt). MS (70 eV): m/z (%) =370 (M+, 2); 341
(1); 298 (3.5); 98 (4.5); 86 (100); 72 (8).
4.1.2.11 2-(4-(methylsulfonyl)phenyl)-3-(pyridin-2-yl)thiazolidin-4-one 6d
Yield: 42,0%; Log P: 1.58; M.p.: 127-129 ºC; 1H NMR δ (CDCl3, 600MHz, ppm, J H-H
3
3
3
4
= Hz); 8.19 (d, 2H, J=8.6, pyridin); 7.85 (d, 2H, J=8.3, Ar); 7.72 (td, 1H, J=7.9, J=1.7,
3
pyridin); 7.49 (d, 2H, J=8.3, Ar); 7.03 (m, 1H, pyridin); 6.91 (s, 1H, H-2); 4.02 (d, 1H,
16

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2
13
2J=16.1, H-5a); 3.84 (d, 1H, J=16.1, H-5b); 3.03 (s, 3H, -SO2CH3). C NMR δ (CDCl3,
150MHz, ppm, J C-F = Hz); 171.0 (C-4); 150.3; 147.8; 147.6; 139.9; 138.0; 127.9 (2C, Ar);
126.7 (2C, Ar); 120.8; 116.4; 62.0 (C-2); 44.4 (-SO2CH3); 33.9 (C-5). MS (70 eV): m/z (%)
=334 (M+, 30); 292 (32); 259 (25); 256 (20); 125 (60); 79 (100).
4.1.2.12 2-(4-(methylsulfonyl)phenyl)-3-(pyridin-2-ylmethyl)thiazolidin-4-one6e
1
Yield: 83.2%; Log P: 1.35; Oil; H NMR δ (CDCl3, 250MHz, ppm, J H-H = Hz); 8.49
(br, 1H, pyridin); 7.91 (d, 2H, 3J=7.3, Ar); 7.64 (q, 1H,3J=7.5, pyridin); 7.48 (d, 2H, 3J=7.5,
Ar); 7.19 (br, 2H, pyridin); 5.83 (s, 1H, H-2);5.04 (d, 1H, 2J=15.0, H-6a); 3.85 (m, 2H, H-5,
H-6b); 3.04 (s, 3H, -SO2CH3). 13C NMR δ (CDCl3, 100MHz, ppm, J C-F = Hz); 171.4 (C-4);
154.9; 149.4; 145.6; 141.0; 136.9; 128.2 (2C, Ar); 128.1 (2C, Ar); 122.8; 122.6; 62.5 (C-2);
48.0 (C-6); 44.3 (-SO2CH3); 32.6 (C-5). MS (70 eV): m/z (%) =273 (M-75; 2); 194 (1.2);
148 (0.7); 134 (1.2); 93 (100); 79 (2).C17H26N2O3S2 (M+H)+: calculated 371.1463; found
371.1475.
4.1.2.13 2-(4-(methylsulfonyl)phenyl)-3-(4-(methylthio)phenyl)thiazolidin-4-one 6f
Yield: 82,6%; Log. P 2.64; M.p.: 217-218 ºC; 1H NMR δ (DMSO, 400MHz, ppm, J H-
3
3
H = Hz); 7.85 (d, 2H, J=8.4, -ArSO2CH3); 7.67 (d, 2H, J=8.4, -ArSO2CH3); 7.31 (d, 2H,
3J=8.7, -ArSCH3); 7.18 (d, 2H, 3J=8.8, -ArSCH3); 6.64 (br, 1H, H-2); 4.07 (dd, 1H, 2J=15.7,
4J=1.5, H-5a); 3.90 (d, 1H, 2J=15.7, H-5b); 3.19 (s, 3H, -SO2CH3); 2.40 (s, 3H, -SCH3). 13C
NMR δ (DMSO, 100MHz, ppm, J C-F = Hz); 170.4 (C-4); 146.1 (Ar); 140.5 (Ar); 136.5 (Ar);
134.0 (Ar); 127.6 (2C, -ArSO2CH3); 127.4 (2C, -ArSO2CH3); 126.0 (2C, -ArSCH3); 125.7
(2C, -ArSCH3); 62.2 (C-2); 43.2 (-SO2CH3); 32.4 (C-5); 14.3 (-SCH3). MS (70 eV): m/z (%)
= 379 (M+, 75); 305 (25); 290 (15); 199 (8); 169 (100); 135 (60).
4.1.2.14
3-(benzo[d][1,3]dioxol-5-yl)-2-(4-(methylsulfonyl)phenyl)thiazolidin-4-one
6g
Yield: 54,0%; Log. P: 1.98; M.p.: 241-243 ºC; 1H NMR δ (DMSO, 600MHz, ppm, J H-
H = Hz); 7.85 (d, 2H, 3J=8.3, Ar); 7.67 (d, 2H, 3J=8.2, Ar); 6.99 (d, 1H, 4J=1.9, Ar); 6.82 (d,
3
4
1H, J=8.3, Ar); 6.77 (dd, 1H,3J=8.1, J=1.9, Ar); 6.52 (s, 1H, H-2);5.97 (d, 2H, J=6.9, -
OCH2O-); 4.02 (d, 1H, 2J=15.7, H-5a); 3.87 (d, 1H, 2J=15.7, H-5b); 3.19 (s, 3H, -SO2CH3).
13C NMR δ (DMSO, 150MHz, ppm, J C-F = Hz); 170.5 (C-4); 147.3 (Ar); 146.0 (Ar); 145.8
(Ar); 140.6 (Ar); 131.0 (Ar); 127.9 (2C, Ar); 127.4 (2C, Ar); 119.4 (Ar); 108.0 (Ar); 107.2
17

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(Ar); 101.5 (-OCH2O-); 62.8 (C-2); 43.2 (-SO2CH3); 32.4 (C-5). MS (70 eV): m/z (%) =377
(M+, 70); 303 (30); 223 (12); 199 (6); 167 (100); 135 (50); 121 (13).
4.2 Animals
Experiments were performed using 1-2 day-old male Wistar rats. These animals
were obtained from the Central Animal Facility of Federal University of Pelotas and were
maintained in temperature controlled environment (23 ± 1 °C), light/dark cycle of 12h, and
the diet both solid as water is provided ad libitum to the animals. The protocol was
approved by the Ethics Committee and Animal Experimentation of Federal University of
Pelotas, under the protocol number CEEA 9219.
4.3 General cell culture procedures
The C6 cell line, of malignant glioblastoma multiform of rats was obtained from
American Type of Culture Collection (ATCC). The cell growth and maintenance was
promoted in low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 0.1%
Fungizone and 100 U/L gentamicin and supplemented with 5% fetal bovine sérum (FBS).
The cells were maintained at 37°C in a humidified a tmosphere with 5% CO2. The culture of
primary astrocyte were prepared as described by Da Frota et al [25]. In summary, the
cortex of neonatal Wistar rats (1-2 days old) were removed and mechanically dissociated
in a salt solution with Ca+2 and Mg+2 free balanced, pH 7.4. After 10 min of centrifugation
(1000 rpm), the pellet was resuspended in DMEM supplemented with 10% FBS. Then, the
cells (3×104) were plated in 96-well plates. After 4 hours, plates were carefully stirred and
washed with phosphate buffered saline (PBS) and the medium was changed. Cultures
were allowed growing up to confluence by 20–25 days and the medium was replaced
every 4 days.
4.4 Cell lines treatment with thiazolidinones
At first, to a screening treatment, all compounds were solubilized in DMSO and
were prepared and tested in DMEM at final concentration of 100 µM, using of standard
TMZ in the same concentration. The glioma cells (C6) were seeded at 5 x 10³ cells/well in
DMEM/5% FBS in 96-well plates. Cultures were exposed to compounds for 48h and 72h
and the control cells were treated with vehicle. Then, the thiazolidinones with the best
results in decreasing tumor cell viability were taken for further tests in various
18

ACCEPTED MANUSCRIPT
concentrations (5, 25, 50, 100 and 250 µM). These tests followed the same protocol
described above.
For cytotoxicity study on a central nervous system cell of primary astrocytes, each
thiazolidinone was dissolved in DMSO and tested at final concentration of 100 and 250
µM. The cells were seeded at 3 x 104 cells/well in DMEM/10% FBS and the culture was
exposed to compounds for 72h, using like negative control vehicle treated cells.
4.5 Cell viability assay
To analyze the anti-tumoral effects of thiazolidinones in C6 glioma cells such as the
cytotoxicity in primary astrocytes cells, it was chosen the method witch assess the cell
viability by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay
.
This method is based on the ability of viable cells to reduce MTT to formazan. The MTT
solution was added in the wells with medium in plates with 96 wells at final concentration
of 0.5 mg/ml. Then, these plates are brought to the incubator for 90 min at 37°C and a
humidified atmosphere with 5% CO2. After, the medium was removed and the formazan
precipitate was shaken with DMSO for 30 min. At 492nm, was measured the optical
density of each well, and the results were expressed as absorbance or percentage of
inhibition, considering the growth of the control group as 100%.
4.6 Propidium iodide assay
The type of cell damage was accessed by fluorescent image analysis of propidium
iodide (PI) uptake. C6 glioma cell line were treated with compounds for 72h and after the
cells were incubated with PI (7.5 ꢀM) for 1h. PI fluorescence was excited at 515–560 nm
using an inverted microscope Olympus IX71 fitted with a standard rhodamine filter. The
images were captured by a digital camera connected to the microscope.
4.7 Statistical analysis
Data were analyzed by using one-way analysis of variance (ANOVA) followed by
Tukey´s post-hoc test for multiple comparisons in the software Graphpad Prism 5.
Furthermore, all data were expressed as mean ± standard error and the differences
between mean values were considered significant when P <0.05.
Supplementary Information
19

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The supplementary information contains the GC/MS and1H and 13C NMR spectra of
all synthesized compounds, besides the HRMS spectra of five compounds.
Acknowledgements
The authors thank UFPel, CAPES and FAPERGS (proc. 11/2068-7) for financial
support. Fellowships also granted to W.C. and R.M.S. by CNPq.
20

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List of captions
Scheme 2. Reagents and synthetic conditions of proposal thiazolidinones.
Figure1. Anti-tumor screening of thiazolidinones (100 µM) in 48h and 72h of exposure.
Bars represent means ± SEM; * represents statistical difference from DMSO control group,
considering P<0.05; **P<0.01; ***P<0.001; # represents statistical difference from TMZ
(100 µM) positive control group, considering P<0.05 (One-way ANOVA followed by post-
hoc Tukey’s test, n=4).
Figure 2. Evaluation of inhibitory effect in different concentrations of thiazolidinones 5b,
5e, 5g and 6e in cell viability. A. C6 cells treatment with different concentrations of 5b, 5e,
5g and 6e in 48h. B. C6 cells treatment with different concentrations of 5b, 5e, 5g and 6e
in 72h. Bars represent means ± SEM; *represents statistical difference from DMSO group,
P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA followed by Tukey’s test, n=4).
Figure 3. Evaluation of treatment of astrocytes cells with proposal thiazolidinones at 100
µM and 250 µM in 72h. *represents statistical difference from DMSO control group,
considering P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA followed by Tukey’s test,
n=3). Bars represent means ± SEM.
Figure 4. Propidium iodide assay of C6 treatment for 72h with thiazolidinones 5b, 5e, 5g
and 6e in 50 and 100 µM. Fluorescence (right panel) and phase contrast (left panel)
microphotographs were taken in 20x magnification.
Figure 5. The atom-numbering for synthesized compounds.
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Thiazolidin-4-ones from 4-(methylthio)benzaldehyde and 4-
(methylsulfonyl)benzaldehyde: Synthesis, antiglioma activity and
cytotoxicity
Highlights:
• Fourteen novel 1,3-thiazolidin-4-ones were efficient obtained;
• Antitumor C6 assay highlights two compounds with activity at 5 µM;
• Three of top four on anti-tumor test were not cytotoxic to normal cell at 250
µM;
• Thiazolidinones as potential anti-GBM effect with safe therapeutic profile.