Synthesis of 1,3-thiazine-2,4-diones with potential anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2013.10.017

2-Amino-1,3-thiazin-4-ones were subjected to acetylation followed by mild acid hydrolysis to give compounds containing the 1,3-thiazine-2,4-dione core. The potential of these S,N-containing heterocycles as antitumor agents against human cancer cell lines,

Full text of DOI:10.1016/j.ejmech.2013.10.017

European Journal of Medicinal Chemistry 70 (2013) 411e418
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 1,3-thiazine-2,4-diones with potential anticancer activity
,
,
Misael Ferreira a 1, Laura Sartori Assunção b 1, Fabíola Branco Filippin-Monteiro b,
Tânia Beatriz Creczynski-Pasa b 1, Marcus Mandolesi Sá a
,
, ,1
*
a Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
b Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Amino-1,3-thiazin-4-ones were subjected to acetylation followed by mild acid hydrolysis to give
compounds containing the 1,3-thiazine-2,4-dione core. The potential of these S,N-containing heterocy-
cles as antitumor agents against human cancer cell lines, among other types, was evaluated. The results
show that phenyl- and naphthyl-substituted thiazinediones presented selective antitumoral activity
against leukemia cells. These compounds caused cell death with DNA fragmentation and the mechanism
of action seems to involve caspase cascade activation, imbalance in intracellular Ca2þ and mitochondrial
metabolism, and/or endoplasmic reticulum stress.
Received 1 September 2013
Received in revised form
3 October 2013
Accepted 7 October 2013
Available online 14 October 2013
Keywords:
Ó 2013 Elsevier Masson SAS. All rights reserved.
1,3-Thiazine-2,4-dione
Anticancer activity
Leukemia
Apoptosis
1. Introduction
pivotal role in regulating gene expression. TZDs, such as the glita-
zones, are recognized for their ability to display antihyperglycemic
Cancer is one of the most prevalent causes of death in several
countries [1]. Despite the huge efforts to implement novel
chemotherapeutic strategies for the treatment of different types of
cancer, these diseases remain one of the major concerns world-
wide. Consequently, there is an urgent need to find unexplored
classes of substances with selective action against cancer cells. The
regulation of the apoptotic pathways associated with cell death is
known as an important approach to understand a great variety of
medical illnesses, including cancer [2,3]. Therefore, the identifica-
tion of apoptosis inducers to combat cancer cells represents an
attractive strategy to the discovery and development of potential
antitumor agents [4,5].
Many heterocyclic compounds containing the SeCeN frame-
work are related to an extensive spectrum of biological activities
[6e8]. In particular, thiazolidine-2,4-diones (TZDs, Fig. 1) are an
important group of S,N-containing heterocycles, which are high-
affinity ligands for peroxisome proliferator-activated receptor
activity and have been widely employed for the treatment of dia-
betes [11e13]. Conversely, recent studies have shown that TZDs also
reduce multiple types of cancer, including breast, colon, lung,
prostate, and stomach [14e18]. However, biological activity related
to 1,3-thiazine-2,4-diones (Fig. 1), the 6-membered heterocycles
homologous to TZD, is less frequently reported [19,20], the only
exception being 5-ethyl-6-phenyl-1,3-thiazine-2,4-dione (Doli-
trone [21]), an anesthetic employed as a pain-killer.
In previous research, we explored cell death pathways, mainly
via apoptosis [22e24], including studies involving the development
of mild and efficient methodologies to construct the 1,3-thiazine
core [25,26] from allylic bromides derived from the MoritaeBay-
liseHillman (MBH) reaction [27]. Consequently, we focused on the
development of a facile strategy for the synthesis of 1,3-thiazine-
2,4-diones and carried out a broad study on their involvement in
apoptotic processes related to antitumor activity.
gamma (PPARg) [9,10], a family of nuclear receptors that play a
2. Results and discussion
2.1. Chemistry
* Corresponding author. Tel.: þ55 48 37216844; fax: þ55 48 37216850.
E-mail address: marcus.sa@ufsc.br (M.M. Sá).
As first authors, M.F. and L.S.A. participated considerably in the parts of the
study related to chemistry and biology, respectively, along with the senior re-
searchers M.M.S. and T.B.C.P.
The synthetic plan employed to prepare the target 1,3-thiazine-
2,4-diones 1 is presented in Scheme 1 and begins with the allylic
bromide 2, a readily available precursor obtained through the
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.017

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M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
reaction as well as the subsequent work-up and purification steps,
affording high-purity products in good isolated yields.
2.2. Cell toxicity
Thiazinediones 1aeh were tested in cancer cell lines that are
widely used as in vitro cancer models, including L1210 (murine
lymphocytic leukemia), CCRF-CEM (human acute lymphoblastic
leukemia), B16F10 (murine melanoma), MDA-MB-231 (human
breast cancer) and a non-tumoral cell line Vero (kidney fibroblast).
The CC50 (concentration of the compound which caused 50% of cell
death) for each cell line was determined by the MTT method fol-
lowed by non-linear regression. In addition, the selectivity index
(SI) was calculated based on the equation:
Fig. 1. Bioactive heterocyclic compounds containing the SeCeN framework.
reaction of
a-methylene-b-hydroxyesters 3 (MBH adducts [27])
with LiBr in acidic medium at room temperature [28e30]. Treat-
ment of the allylic bromides 2 with thiourea in a 3:1 mixture of
acetone:water at room temperature followed by the addition of a
mild aqueous base to the pre-formed isothiouronium salts 4 gave
high yields of the corresponding 2-amino-1,3-thiazin-4-ones 5 as
insoluble solids [25,26].
CC50 non tumoral cells
SI ¼
CC50 tumoral cells
Attempts to directly transform the key 2-aminothiazin-4-one 5a
into thiazine-2,4-dione 1a by hydrolysis in acidic medium [31,32]
led to modest yields (up to 30%). In view of this shortcoming, a
two-step (one-pot) method had to be developed, which consisted of
the initial acetylation of 2-aminothiazin-4-one 5a followed by mild
hydrolysis of the acetylated intermediate. The 2-aminothiazin-4-
one 5a was then acetylated using acetic anhydride in ethanol to
give an approximately 1:1 mixture of two (out of four) possible
acetylated isomers 6e9 (Scheme 1), as noted by the two singlets
corresponding to distinct acetyl groups at 2.03 and 2.27 ppm in the
1H NMR spectrum of the crude reaction. All modifications to the
reaction parameters led to mixtures of acetylated products that
underwent slow hydrolysis after prolonged exposure to air, while
attempts to obtain an isolated product by column chromatography
in silica gel failed due to extensive decomposition. On the other
hand, the simple addition of aqueous HCl to the pre-formed
mixture of acetylated products (6, 7, 8, and/or 9) promoted clean
and convergent hydrolysis to 5-benzylidene-1,3-thiazine-2,4-dione
1a in 82% yield (Table 1).
The results are summarized in Table 2. Compounds 1a and 1g
demonstrated toxic effect at lower concentrations not only against
leukemia (L1210) cells (35 and 28
melanoma (B16F10) cells (50 and 45
m
M, respectively) but also toward
M, respectively). In addition,
m
higher selectivity (SI ꢀ 2.0) was observed in both cases, compared
with the other screened thiazinediones. Because compounds 1a
and 1g showed high selectivity toward leukemia and melanoma,
among other cancer cell lines, cell cycle analysis by flow cytometry
was performed to evaluate possible alterations in these cells. Cells
were treated with 35 mM of 1a and 28 mM of 1g for 24 h. As shown
in Fig. 2, compounds 1a and 1g caused cell fragmentation in leu-
kemia cells, but no alterations were seen in melanoma cells. In the
light of these findings, the subsequent investigation on the mech-
anism of action was carried out using leukemia cells.
2.2.1. Mechanism of action
To investigate whether the observed cell death caused by 1a and
1g in leukemia cells was due to necrosis or apoptosis, quantitative
and qualitative evaluations were carried out. Initially, a morpho-
logical evaluation employing fluorescence microscopy with
double-staining acridine orange/ethidium bromide was performed.
Acridine orange (AO) easily permeates the cell membrane and re-
sults in a green fluorescence of both the nucleus and cytoplasm.
Ethidium bromide (EB), however, does not permeate the
This sequential acetylation/hydrolysis protocol was then
extended to other thiazinones 5 with the exclusive formation of the
expected 1,3-thiazine-2,4-diones 1 (Scheme 1 and Table 1; see also
the Supplementary information). One of the most remarkable as-
pects of this methodology is its simplicity in terms of setting up the
Scheme 1. Synthesis of 1,3-thiazine-2,4-diones.

M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
413
Table 1
compounds 1a and 1g induced apoptosis. In fact, 26% and 14% of
cells underwent late apoptosis when treated with 1a and 1g,
respectively. Moreover, apoptotic cells were observed in approxi-
mately 14% of cells treated with both compounds (Fig. 4).
One-pot synthesis of 1,3-thiazine-2,4-diones 1 from 2-aminothiazin-4-ones 5.
Compound
1,3-Thiazine-2,4-dione
Time (h)a
Yield (%)b
O
1a
NH
2
82
2.2.2. Mitochondrial membrane potential (MMP) measurement and
ROS generation
S
O
Substances which can compromise the integrity of mitochon-
dria and stimulate the release of factors that induce cell apoptosis
may induce cell death and thus prevent the proliferation of tumor
cells [35,36]. The presence of permeability transition pores (PTP) in
mitochondria leads to a loss of inner transmembrane potential
O
S
NH
1b
1c
1d
1e
1f
3
4
4
4
4
68
58
77
80
75
H3CO
O
O
S
(DJm) [37]. This phenomenon can occur due to the release of
O
O
NH
proapoptotic mitochondrial proteins, such as those from the Bcl2
family, and/or by Ca2þ-induced PTP opening, resulting in a release
of cytochrome c and other proapoptotic factors that might be
associated with osmotic swelling, leading to a high concentration of
proteins in the matrix, which could culminate in the rupture of the
mitochondrial membrane [38]. The membrane-permeant JC-1 dye
(5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolcarbocianyne
iodide) is widely used in apoptosis studies to monitor mitochon-
drial activity. Thus, leukemia cells were treated with 1a and 1g and
the mitochondrial membrane potential (MMP) was evaluated.
When the cells were exposed to the compounds (CC50), the MMP
was not affected; however, an increase in the concentration of both
compounds from CC50 to CC70 caused a decrease in the membrane
potential (Fig. 5A).
O
O
NH
Cl
Cl
S
O
O
S
NH
O
O
Cl
NH
Cl
S
O
O
The mitochondrial activity is also closely related to the reactive
oxygen species (ROS), which are by-products of metabolic reactions
in aerobic cells that occur in response to various stimuli. Their
presence can lead to oxidative stress resulting in cell destruction by
necrosis. However, the apoptotic process is triggered when ROS
derive from mitochondria, the major organelle involved in oxida-
tive metabolism imbalance. ROS participate in both the late and
early stages of apoptosis, for instance, in the regulation of apoptosis
preceding mitochondrial membrane permeabilization, making it
difficult to determine whether the accumulation of ROS is the cause
of the process or a side effect of other alterations [39]. ROS pro-
duction can also be a consequence of endoplasmic reticulum stress
(ERS), which leads to a Ca2þ-induced depolarization of the inner
mitochondrial membrane [40]. Due to the extensive participation
of ROS in the apoptotic process, their production was evaluated in
cells treated with 1a and 1g and analyzed quantitatively using the
DCF-DA fluorescent probe. Hydrogen peroxide was used as a pos-
itive control. For both compounds, the ROS production increased in
cells treated with the CC50 concentration. Likewise, when the cells
were exposed to the CC70 concentration of these compounds, the
ROS generation was increased 2-fold for 1a and 3-fold for 1g when
compared to the control cells (Fig. 5B).
1g
NH
2
2
78
85
S
O
O
1h
NH
S
O
a
Time (h) refers to the total reaction time for the one-pot procedure, consisting of
first treating the thiazinone 5 with acetic anhydride in EtOH for 1 h at 25 ꢁC followed
by adding 1 M HCl to the reaction with continued stirring at 25 ꢁC for an additional
1e3 h.
b
Isolated yields from thiazinones 5.
cytoplasmic membrane in viable cells, unless the integrity is
compromised, when it intercalates into the DNA and stains it red.
Necrotic cells display
a structurally normal orange nucleus,
apoptotic cells have a green nucleus with fragmented chromatin,
and cells in late apoptosis have a fragmented orange chromatin
[33,34]. As shown in Fig. 3, compounds 1a and 1g caused DNA
fragmentation, indicative of late apoptosis after treatment with the
CC50 concentration of each compound for 24 h.
Subsequently, a flow cytometry assay using propidium iodide, a
DNA label, and Annexin-V-FITC, a fluorescent probe that binds to
phosphatidyl serine of apoptotic cells, confirmed that the
Table 2
CC50 values of compounds against different cancer cell lines.
Compound
CC50
Vero
(m
M)a
Selectivity index (SI)b
L1210
CCRF-CEM
B16F10
MDA-MB-231
Vero/L1210
Vero/CCRF-CEM
Vero/B16F10
Vero/MDA-MB-231
1a
1b
1c
1d
1e
1f
100
100
55
100
60
41
96
100
35
100
63
60
27
30
28
100
e
50
100
48
100
48
32
45
100
74
63
96
98
52
35
93
100
2.6
1.0
0.9
1.7
2.2
1.4
3.4
1.0
e
2.0
1.0
1.1
1.0
1.3
1.3
2.1
1.0
1.4
1.6
0.6
1.0
1.2
1.2
1.0
1.0
e
e
e
e
96
49
40
64
100
1.0
1.2
1.0
1.5
1.0
1g
1h
a
Data were expressed as mean ꢂ SD (n ¼ 3).
b
Defined as the ratio between CC50 of non tumoral cell and tumoral cell.

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M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
Fig. 2. Cell cycle of Leukemia (L1210) (A) and melanoma (B16F10) (B) cell lines after 24-h treatment with 1a (35 mM) and 1g (28 mM). G0/G1, G2/M and S indicate the cell phase, and
sub-G1 DNA content refers to the proportion of fragmented cells. Each phase was calculated using the WinMDI 2.9 program. **p < 0.01, *p < 0.05, n ¼ 3.
Fig. 3. Fluorescence microscopy with double staining acridine orange/ethidium bromide of leukemia cells (L1210) without treatment and treated with CC50 concentration of
compounds 1a and 1g. Yellow arrows indicate cell fragmentation and white arrows indicate necrosis (magnification at 400ꢃ). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
2.2.3. Caspases and intracellular calcium measurements
However, treatment with compound 1g for 12 h induced the
caspase-3 activity in comparable magnitude than that observed for
compound 1a (Fig. 6B), leading to the consideration that it takes
more time to induce caspase-3 activation when cells are treated
with compound 1g. Interestingly, a caspase-3 inhibition was
observed when cells were treated with compound 1g for a period of
4 h. Also, there was a significant caspase-8 inhibition caused by
both compounds 1a and 1g after 4 h, but after 12 h this phenom-
enon no longer persists, leading to the conclusion that the decrease
in caspase-8 activity consists in a transitory event.
The apoptotic process is mediated by a family of cysteine pro-
teases known as caspases, which can be activated by extrinsic and/
or intrinsic pathways to cleave their proteic substrates and release
aspartatic residues [35]. To evaluate the participation of caspases in
the cell death process induced by thiazinediones 1a and 1g, leu-
kemia cells were treated with the CC50 concentration of each
compound, and the production of a fluorogenic substrate derived
from caspase-3, -8, -9 and -12 activity was monitored. As shown in
Fig. 6A, a significant increase in caspase-3 and -12 activities was
observed after treatment with compound 1a for 4 h. Compound 1g
increased caspase-12 but not caspase-3 activity after 4-h treatment.
The activation and inactivation of caspases are regulated by
various proteins and ions, including Ca2þ [41]. The Ca2þ ion not only
Fig. 4. Flow cytometry with Annexin-V-FITC and propidium iodide of leukemia cells (L1210) to evaluate the cell death profile after 24-h treatment. Q1 cells were considered necrotic
cells, Q2 were considered cells in late apoptosis, Q3 live cells and Q4 apoptotic cells. Cells without treatment were considered control group.

M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
415
Fig. 5. Effect of CC50 and CC70 concentrations of compounds 1a and 1g on the mitochondrial membrane potential of leukemia cells (L1210) using JC-1 probe. Cells were incubated for
4 h with 1 M FCCP as positive control. The decrease of red/green ratio indicates a decrease in mitochondrial membrane potential, and the results are expressed as percentage
m
control cells (100%) (A). Evaluation of ROS generation induced by CC50 and CC70 concentrations of compounds 1a and 1g on L1210 cells after 4-h treatment. H2O2 was used as
positive control. Free radical formation was followed using DCFH-DA as described in the Experimental section. The results were expressed in units of fluorescence in comparison to
control group (cells without treatment) and normalized by the protein concentration (B). ***p < 0.001, **p < 0.01, *p < 0.05, n ¼ 3.
Fig. 6. Quantification of caspase-3, -8, -9 and -12 in L1210. Cells were treated with CC50 concentrations of compounds 1a and 1g. Caspase activity was presented as fluorescence unit
in comparison to control group (cells without treatment) after 4-h (A) and 12-h treatment (B). Evaluation of intracellular calcium changes after 4-h treatment with the CC50 of
compounds 1a and 1g in L1210 cells using fluorescent probe Fluo-3-AM. Calcium ionophore A23187 was used as positive control. Results are expressed as a ratio of Fluo-3 AM/
protein content fluorescence compared with control cells (without treatment) (C). ***p < 0.001, **p < 0.01, *p < 0.05, n ¼ 3.
participates in cell survival but also triggers proapoptotic events
when there is interference in the ion uptake into intracellular pools
such as the endoplasmic reticulum (ER) and mitochondria [35]. At
physiological levels, the Ca2þ released from the ER during cell
activation is taken up by mitochondria to enhance the metabolite
flow across the outer membrane and promotes oxidative phos-
phorylation increasing the ATP production [42]. Mobilizations of
the intracellular Ca2þ ([Ca2þ]i) store, for instance depletion or al-
terations in the transport systems, can trigger an ER stress leading
to the release of procaspase-12 from the ER membrane. Once
activated, caspase-12 may induce the activation of effector caspases
and eventually the apoptosis [38]. After 4-h treatment with the
CC50 concentration of compounds 1a and 1g, changes in intracel-
lular calcium homeostasis were observed, as shown in Fig. 6C.
The involvement of calcium imbalance besides caspase-3 and 12
activation in the process of cell death induced by compounds 1a
and 1g raises the possibility that the mechanism of action is related
to the calcium-calpain-caspase-12-caspase-3 cascade [43]. The
development of new drugs for cancer therapy is based on the po-
tential of the compounds to interrupt cell proliferation and/or to
trigger cell death. Targeting the apoptotic pathways of cancer cells
has become an attractive strategy helping the discovery of new
drugs to circumvent the side effects (the main limitation of the
cancer therapies), as well as the occurrence of drug resistance.
death with DNA fragmentation and the mechanism of action seems
to involve caspase cascade activation, imbalance in intracellular
Ca2þ and mitochondrial metabolism and/or ER stress.
4. Experimental section
4.1. Chemistry
4.1.1. General considerations
All chemicals were of reagent grade and were used as received.
Melting points were determined using a Microquímica MQPF301
hot-plate apparatus and are uncorrected. Infrared spectra were ac-
quired with a PerkineElmer FT-IR 1600 spectrometer (range 4000e
400 cmꢄ1) using KBr for solids samples. 1H NMR (400 MHz) and 13
C
NMR (100 MHz, fully decoupled) spectra were recorded with a
Varian AS-400 spectrometer. Samples were prepared in an appro-
priate deuterated solvent (CDCl3 or DMSO-d6). Chemical shifts are
reported in parts per million (ppm, d) relative to TMS at 0.00 ppm or
solvent (CDCl3 at 7.26 ppm or DMSO-d6 at 2.50 ppm for 1H NMR, and
CDCl3 at 77.16 ppm or DMSO-d6 at 39.52 ppm for 13C NMR) as the
internal standard. Coupling constants (J) are measured in Hertz (Hz)
and coupling patterns are designated as s (singlet); d (doublet); dd
(doublet of doublets); m (multiplet); brs (broad signal). Elemental
analyses were conducted in a CHNSeO Carlo Erba EA-1110 analyzer.
The 2-amino-1,3-thiazin-4-ones 5aeh were prepared and purified
according to the previously described methods [25,26].
3. Conclusion
In summary, thiazinediones 1a and 1g, which were readily
prepared using simple reagents under mild conditions, presented
selective antitumoral activity against leukemia cells. Phenyl- and
naphthyl-substituted 1,3-thiazine-2,4-diones 1a and 1g caused cell
4.1.2. Typical procedure for the synthesis of 1,3-thiazine-2,4-diones
1
To a stirred suspension of 1,3-thiazin-4-one 5 (1.0 mmol) in
3.0 mL of ethanol at 25 ꢁC was added 3.0 mmol of acetic anhydride

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M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
and the reaction mixture was stirred for 1 h. Then, 1 M HCl (1.0 mL)
was added and the final mixture was further stirred at room tem-
perature. After 1e3 h (Table 1), the reaction was diluted with
CH2Cl2 and H2O and the organic extract was separated, washed
with H2O and concentrated under reduced pressure. The resulting
solid was crushed with ethyl ether and filtered to give the corre-
sponding 1,3-thiazine-2,4-dione 1.
C11H7Cl2NO2S (%): C, 45.85; H, 2.45; N, 4.86; S, 11.13. Found: C,
45.56; H, 2.62; N, 4.77; S, 10.71.
4.1.2.7. (5Z)-5-[(Naphthalen-2-yl)methylene]-5,6-dihydro-1,3-
thiazine-2,4-dione (1g). Yellow solid, mp 184.0e186.0 ꢁC. IR (KBr)
nmax/cmꢄ1: 3468, 3169, 3055, 2835, 1685, 1626, 1606, 1352, 1195,
1159. 1H NMR (400 MHz, DMSO-d6):
d
4.31 (s, 2H), 7.53e7.60 (m,
3H), 7.79 (s, 1H), 7.93e7.99 (m, 3H), 8.05 (s, 1H), 11.55 (brs, 1H). 13
NMR (100 MHz, DMSO-d6): 25.4 (CH2), 125.1 (C), 126.8 (CH), 126.9
C
4.1.2.1. (5Z)-5-Benzylidene-5,6-dihydro-1,3-thiazine-2,4-dione (1a).
White solid, mp 158.5e160.0 ꢁC. IR (KBr) nmax/cmꢄ1: 3445, 3156,
3046, 2855, 1695, 1666, 1630, 1335, 1252, 1187, 1181, 1158. 1H
d
(CH), 127.4 (CH), 127.6 (CH), 128.4 (CH), 128.5 (CH), 129.7 (CH), 131.4
(C), 132.7 (C), 133.0 (C), 138.8 (]CH), 166.7 (C), 168.2 (C). Anal.
Calcd. for C11H9NO2S: C, 66.89; H, 4.12; N, 5.20; S, 11.91. Found: C,
66.51; H, 4.25; N, 5.11; S, 11.34.
NMR (400 MHz, CDCl3):
d
4.07 (s, 2H), 7.34e7.48 (m, 5H), 7.89 (s,
26.0 (CH2),
1H), 8.81 (brs, 1H). 13C NMR (100 MHz, CDCl3):
d
123.6 (C), 129.1 (2 ꢃ CH), 129.5 (2 ꢃ CH), 129.9 (CH), 133.9 (C),
141.9 (]CH), 166.4 (C), 168.0 (C). Anal. Calcd. for C11H9NO2S (%):
C, 60.26; H, 4.14; N, 6.39; S, 14.62. Found: C, 60.55; H, 4.20; N,
6.35; S, 14.74.
4.1.2.8. (5Z)-5,6-Dihydro-5-[(E)-3-phenyl-2-propenylidene]-1,3-
thiazine-2,4-dione (1h). Yellow solid, mp 208.0e210.0 ꢁC. IR (KBr)
nmax/cmꢄ1: 3446, 3178, 3094, 3037, 2822, 1691, 1657, 1611, 1590,
1428, 1344, 1198, 1162. 1H NMR (400 MHz, DMSO-d6):
d
4.26 (s, 2H),
7.19 (d, J ¼ 15.2 Hz, 1H), 7.28e7.48 (m, 5H), 7.65 (d, J ¼ 7.2 Hz, 2H),
11.37 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
24.6 (CH2), 123.1
4.1.2.2. (5Z)-5,6-Dihydro-5-(4-methoxybenzylidene)-1,3-thiazine-
2,4-dione (1b). White solid, mp 164.0e165.0 ꢁC. IR (KBr) nmax/cmꢄ1
:
d
3453, 3169, 3063, 2837, 1685, 1600, 1508, 1342, 1304, 1257, 1172. 1H
(]CH), 123.2 (C), 127.7 (2 ꢃ CH), 128.9 (2 ꢃ CH), 129.4 (CH), 136.0
NMR (400 MHz, CDCl3):
d 3.85 (s, 3H), 4.10 (s, 2H), 6.97 (d,
(C), 138.3 (]CH), 142.3 (]CH), 166.6 (C), 168.4 (C). Anal. Calcd. for
J ¼ 8.7 Hz, 2H), 7.33 (d, J ¼ 8.7 Hz, 2H), 7.83 (s, 1H), 8.55 (brs, 1H). 13
C
C13H11NO2S (%): C, 63.65; H, 4.52; N, 5.71; S, 13.07. Found: C, 63.21;
H, 4.51; N, 5.77; S, 12.94.
NMR (100 MHz, CDCl3):
d
26.1 (CH2), 55.5 (OCH3), 114.6 (2 ꢃ CH),
121.1 (C), 126.4 (C), 131.7 (2 ꢃ CH), 141.9 (]CH), 161.0 (C), 166.6 (C),
168.0 (C). Anal. Calcd. for C12H11NO3S (%): C, 57.82; H, 4.45; N, 5.62;
S, 12.68. Found: C, 57.59; H, 4.45; N, 5.51; S, 13.11.
4.2. Biological activity
4.2.1. Materials
4.1.2.3. (5Z)-5,6-Dihydro-5-(3,4-methylenedioxybenzylidene)-1,3-
thiazine-2,4-dione (1c). White solid, mp 199.0e200.0 ꢁC. IR (KBr)
nmax/cmꢄ1: 3442, 3272, 3053, 2906, 1697, 1657, 1628, 1594, 1497,
The cell culture media and fetal bovine serum were purchased
from Cultilab. The antibiotics penicillin/streptomycin were supplied
by GIBCO. The JC-1 probe (5,50,6,60-tetrachloro-1,10,3,30-tetrae-
thylbenzimidazolcarbocyanine iodide) and DCFH-DA (20,70-
dichlorofluorescein diacetate) were purchased from Invitrogen.
Propidium iodide was supplied by Milipore. Dimethyl sulfoxide
(DMSO) was purchased from Merck and all other reagents were
purchased from SigmaeAldrich.
1348, 1321, 1247, 1193. 1H NMR (400 MHz, DMSO-d6):
d
4.19 (s,
2H), 6.07 (s, 2H), 6.98e7.10 (m, 3H), 7.55 (s, 1H), 11.43 (brs, 1H). 13
NMR (100 MHz, DMSO-d6): 25.3 (CH2), 101.7 (CH2), 108.8 (CH),
C
d
109.6 (CH), 123.0 (C), 125.3 (CH), 127.8 (C), 138.9 (]CH), 147.8 (C),
148.5 (C), 166.8 (C), 168.2 (C). Anal. Calcd. for C12H9NO4S (%): C,
54.75; H, 3.45; N, 5.32; S, 12.18. Found: C, 54.77; H, 3.46; N, 5.29; S,
12.54.
4.2.2. Cell lines and culture conditions
Cell lines were purchased from the Rio de Janeiro Cell Bank. The
cells were cultured in RPMI-1640 or DEMEM medium, supple-
mented with 1.5 g/L sodium bicarbonate, 10 mM HEPES, pH 7.4,
4.1.2.4. (5Z)-5-(4-Chlorobenzylidene)-5,6-dihydro-1,3-thiazine-2,4-
dione (1d). White solid, mp 211.0e213.0 ꢁC. IR (KBr) nmax/cmꢄ1
:
3449, 3165, 3104, 3038, 2844, 1685, 1655, 1602, 1489, 1408, 1328,
1199. 1H NMR (400 MHz, DMSO-d6):
4.17 (s, 2H), 7.50 (m, 4H), 7.61
(s, 1H), 11.54 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
25.1 (CH2),
125.6 (C),128.9 (2 ꢃ CH),131.6 (2 ꢃ CH),132.8 (C),134.1 (C),137.5 (]
CH),166.5 (C),168.1 (C). Anal. Calcd. for C11H8ClNO2S (%): C, 52.08; H,
3.18; N, 5.52; S, 12.64. Found: C, 51.77; H, 3.32; N, 5.44; S, 12.46.
100 U/mL penicillin G, 100 mg/mL streptomycin and 10% fetal calf
d
serum at 37 ꢁC in a humidified atmosphere consisting of 95% air and
5% CO2. Cells were passaged approximately twice a week and cul-
tures with greater than 95% of viable cells in trypan-blue exclusion
tests were used for the experiments.
d
4.2.3. Viability assay
4.1.2.5. (5Z)-5-(2-Chlorobenzylidene)-5,6-dihydro-1,3-thiazine-2,4-
The MTT method was used to determine the cell viability [44].
Adherent cells (1 ꢃ 104/well) and suspended cells (1 ꢃ 105/well)
were seeded in 96-well plates and incubated for 24 h with
dione (1e). White solid, mp 156.0e157.0 ꢁC. IR (KBr) nmax/cmꢄ1
:
3455, 3160, 3064, 2853, 1688, 1669, 1620, 1470, 1338, 1188. 1H NMR
(400 MHz, DMSO-d6): 4.06 (s, 2H), 7.40e7.60 (m, 4H), 7.62 (s, 1H),
11.62 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
25.2 (CH2),127.2 (C),
d
increasing concentrations of the compounds, ranging from 15
mM
d
to 100 M. For the control group, cells were incubated without
m
127.6 (CH),129.8 (CH),130.8 (CH),131.1 (CH),132.1 (C),133.4 (C),135.0
(]CH), 166.2 (C), 168.1 (C). Anal. Calcd. for C11H8ClNO2S: C, 52.08; H,
3.18; N, 5.52; S, 12.64. Found: C, 52.04; H, 3.25; N, 5.49; S, 12.66.
treatment. After incubation, the old culture medium was replaced
with fresh culture medium with 5 mg/mL of MTT, followed by in-
cubation for 1e4 h at 37 ꢁC. MTT-formazan crystals were dissolved
in 100 mL of DMSO and the absorbance was measured at 540 nm
4.1.2.6. (5Z)-5-(2,4-Dichlorobenzylidene)-5,6-dihydro-1,3-thiazine-
using a micro-well system reader. The CC50 values were calculated
through a Hill concentrationeresponse curve.
2,4-dione (1f). White solid, mp 176.0e177.5 ꢁC. IR (KBr) nmax/cmꢄ1
:
3436, 3167, 3055, 3005, 2859, 1693, 1669, 1636, 1583, 1468, 1360,
1334, 1194, 1170. 1H NMR (400 MHz, DMSO-d6):
d
4.05 (s, 2H), 7.43
4.2.4. Morphological evaluation assay
(d, J ¼ 8.5 Hz, 1H), 7.51 (dd, J ¼ 2.0 and 8.5 Hz, 1H), 7.55 (s, 1H), 7.77
A double staining method, with ethidium bromide (3,8-
(d, J ¼ 2.0 Hz, 1H), 11.64 (brs, 1H). 13C NMR (100 MHz, DMSO-d6):
diamino-5-ethyl-6-phenylphenanthridinium bromide) and acri-
dine orange (3,6-dimethylaminoacridine), was used to evaluate the
morphological alterations and identify the cell death type (necrosis
d
25.2 (CH2), 127.8 (CH), 127.9 (C), 129.4 (CH), 131.2 (C), 132.1 (CH),
133.9 (]CH), 134.4 (C), 134.8 (C), 166.1 (C), 168.1 (C). Anal. Calcd. for

M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
417
versus apoptosis). The cells (1 ꢃ 106/well) were seeded in 12-well
plates and incubated with the CC50 concentrations of each com-
pound 1aeh for 24 h. The control group was incubated with pure
growth medium. After incubation, cells were centrifuged at 400 g at
room temperature for 10 min. After removal of the growth medium,
order to analyze the results, which were subsequently normalized
as a percentage of the untreated control (100%).
4.2.8. Determination of caspase-3, -8, -9 and -12 activity
Caspase-3, -8, -9 and -12 activity was monitored through the
production of fluorescent substrate. Briefly, L1210 cells were seeded
in a 6-well plate (3 ꢃ 106 cells/well) and treated with CC50 con-
centrations of 1a and 1g for 4 and 12 h. The cells were washed
cells were resuspended in 500
ethidium bromide and 0.6 g of acridine orange. After 2 min, cells
were centrifuged again, resuspended in 500 L of pure PBS and
mL of PBS containing 2 mg of
m
m
examined under a fluorescence microscope (Nikon, Eclipse TS100).
with PBS and lysed in 100 mL of lysis buffer containing 50 mM
HEPES pH 7.4, 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate (CHAPS), 5 mM dithiothreitol (DTT), 1 mM phe-
4.2.5. Flow cytometry analysis
To analyze the cell cycle of cells treated with 1,3-thiazine-2,4-
nylmethylsulfonyl fluoride (PMSF), 1 mg/mL pepstatin A, 1 mg/mL
diones 1, flow cytometry was used following
a
previously
leupeptin, and 5 mg/mL aprotinin, homogeneized in a vortex and
described method [45]. Briefly, the cells (1 ꢃ 106/well) were incu-
bated with the CC50 concentrations of each 1,3-thiazine-2,4-dione 1
for 24 h, in a 12-well plate. After incubation, the cells were
centrifuged at 400 g at room temperature for 10 min. The cells were
washed with 1 mL of PBS and centrifuged again. The supernatant
incubated for 20 min at 4 ꢁC. After cell lysis, the samples were
centrifuged at 14,000 g for 15 min at 4 ꢁC and homogenates were
obtained in the supernatant. The extract (50 mL) was then added to
a reaction medium containing 25 mM HEPES pH 7.4, 1% CHAPS,
1 mM EDTA, 10% sucrose, 3 mM DTT and supplemented with the
was discarded and cells were fixed with 200
m
L of 70% ethanol for
corresponding substrate for each caspase (caspase-3: 20
DEVD-AMC; caspase-8: 100 M Ac-IEDT-AMC; caspase-9: 100
Ac-LEDH-AFC; caspase-12: 50 M Ac-ATAD-AFC). Cleavage of the
fluorogenic substrate was detected spectrofluorimetrically (Per-
kineElmer LS55, Boston, MA, USA) after incubation for 2 h at 37 ꢁC,
using excitation and emission wavelengths of 380 and 460 nm,
respectively [47]. The results were expressed in arbitrary units of
fluorescence, considering the activity of the control as one unit.
mM Ac-
30 min at 4 ꢁC. PBS (1 mL) with 2% of BSA was then added and
centrifuged for 10 min at 400 g. The supernatants were removed
and the cells were permeabilized with lysis buffer (0.1% Triton-X in
m
mM
m
PBS) and 0.5
mL of RNase (100
mg/mL). The DNA content was stained
with propidium iodide (20
mg/mL) and analyzed using a FACSCanto
flow cytometer (Becton Dickinson). The cell population in each
phase of the cell cycle was determined using WinMDI 2.9 software.
To evaluate the occurrence of apoptosis, the cells (1 ꢃ 106/well)
were treated with the CC50 concentration of 1,3-thiazine-2,4-
diones 1 for 24 h, in a 12-well plate, centrifuged at 400 g at 4 ꢁC
for 10 min, washed twice with 1 mL of cold PBS, resuspended in
4.2.9. Intracellular calcium
To measure changes in the intracellular calcium, the fluorescent
probe Fluo-3-AM was used. L1210 cells were seeded in a 24-well plate
(1 ꢃ 106 cells/well), treated for 4 h with the CC50 concentration of
compounds 1a and 1g and centrifuged at 400 g for 10 min. The old
binding buffer (Millipore) and incubated with 2.5
mL Annexin-V-
FITC and 5 L of 20 g/mL propidium iodide solution for 15 min.
m
m
Cell death was analyzed using a FACSCanto flow cytometer (Becton
Dickinson) and WinMDI 2.9 software. The percentage of apoptosis/
necrosis induced by the treatments was compared to that of the
control group (cells without treatment).
medium was removed and a solution of 300
m
L of HBBS with 0.02%
Pluronic F127 and Fluo-3 AM (3
m
M) was added to the cells which
were incubated for 20 min at 37 ꢁC. After incubation, 1.2 mL of HBSS
with 1% FBS was added followed by incubation for 40 min at 37 ꢁC.
Cells were then centrifuged at 400 g for 10 min for supernatant
removal, washed three times with HEPES buffer containing 0.1% of
4.2.6. Mitochondrial membrane potential
To evaluate the mitochondrial membrane potential of the cells
treated with the CC50 concentration of 1,3-thiazine-2,4-diones 1, the
lipophilic, cationic, fluorescentprobeJC-1 wasused. The cells (1 ꢃ106/
well) were seeded in a 12-well plate and incubated for 4 h with the
compounds. After incubation, the probe was added and left for 20 min
at 37 ꢁC (5% CO2). The cells were then washed twice with 1 mL of PBS
BSA at 37 ꢁC, and resuspended in 200
mL of PBS for subsequent
measurement of the fluorescence in a spectrofluorimeter (Perkine
Elmer LS55) at an excitation of 488 nm and emission of 526 nm. The
protein content was measured at an excitation of 280 nm and emis-
sion of 340 nm. The calcium ionophore A23187 was used as a positive
control and results were presented as the ratio of Fluo-3 AM/protein
content fluorescence and compared with the control cells (without
treatment), which were considered to have no calcium efflux.
and resuspended in 500 mL of PBS for subsequent measurement of the
fluorescence in a spectrofluorimeter (PerkineElmer LS55) with an
excitation of 488 nm and emission of 590 nm and 527 nm for red and
green fluorescence, respectively. Results were presented as the 590/
527 fluorescence ratio and compared with the control cells (without
treatment), which were considered to have 100% mitochondrial
membrane potential. FCCP, an electron transport chain uncoupler,
Acknowledgments
The authors wish to thank the Central de Análises (Departa-
mento de Química, UFSC, Florianópolis) for spectroscopic analysis.
M.F. and L.S.A. are grateful to CAPES (Coordenação de Aperfeiçoa-
mento de Pessoal de Nível Superior) and CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico) for fellowships.
T.B.C.P. and M.M.S. are grateful to CNPq for research fellowships.
Financial support from INCT-Catalysis/CNPq (Instituto Nacional de
Catálise de Sistemas Nanoestruturados, Brazil) is also gratefully
acknowledged.
was used as the positive control at a concentration of 1 mM.
4.2.7. ROS generation
The production of intracellular reactive species was evaluated
using the DCFH-DA probe. Cells were seeded in 24-well plates
(1 ꢃ106 cells/well) and treated for 4 h with 35
mM and 42 mM of 1a
mM and 33.6 mM of 1g. As positive controls, cells were
and 28
treated with H2O2 for 1 h. At the end of the incubation time, cells
were stained with 10
M DCFH-DA for 30 min at 37 ꢁC, washed four
times with 1 mL of PBS and resuspended in 500 L of PBS. The
m
Appendix A. Supplementary data
m
evaluation was performed by quantification of the green fluores-
cence in a spectrofluorimeter (PerkineElmer LS55, Boston, MA,
USA) at an excitation of 480 nm and emission of 520 nm [46]. An
analytical curve was constructed using a standard DCF solution in
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.10.
017. These data include MOL files and InChiKeys of the most
important compounds described in this article.

418
M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418
[24] A.L.F. Navarini, L.D. Chiaradia, A. Mascarello, M. Fritzen, R.J. Nunes, R.A. Yunes,
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