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 b

Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil 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: Received 1 September 2013 Received in revised form 3 October 2013 Accepted 7 October 2013 Available online 14 October 2013

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, 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. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: 1,3-Thiazine-2,4-dione Anticancer activity Leukemia Apoptosis

1. Introduction 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 worldwide. 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 identification 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 framework 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 highaffinity ligands for peroxisome proliferator-activated receptor gamma (PPARg) [9,10], a family of nuclear receptors that play a

pivotal role in regulating gene expression. TZDs, such as the glitazones, are recognized for their ability to display antihyperglycemic activity and have been widely employed for the treatment of diabetes [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 (Dolitrone [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 MoritaeBayliseHillman (MBH) reaction [27]. Consequently, we focused on the development of a facile strategy for the synthesis of 1,3-thiazine2,4-diones and carried out a broad study on their involvement in apoptotic processes related to antitumor activity.

2. Results and discussion

* Corresponding author. Tel.: þ55 48 37216844; fax: þ55 48 37216850. E-mail address: [email protected] (M.M. Sá). 1 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 researchers M.M.S. and T.B.C.P. 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

2.1. Chemistry The synthetic plan employed to prepare the target 1,3-thiazine2,4-diones 1 is presented in Scheme 1 and begins with the allylic bromide 2, a readily available precursor obtained through the

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reaction as well as the subsequent work-up and purification steps, affording high-purity products in good isolated yields. 2.2. Cell toxicity

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]. Treatment 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]. 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-4one 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 1 H 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). 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 aspects of this methodology is its simplicity in terms of setting up the

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 followed by non-linear regression. In addition, the selectivity index (SI) was calculated based on the equation:

SI ¼

CC50 non tumoral cells CC50 tumoral cells

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 mM, respectively) but also toward melanoma (B16F10) cells (50 and 45 mM, respectively). In addition, 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 leukemia cells, but no alterations were seen in melanoma cells. In the light of these findings, the subsequent investigation on the mechanism 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 morphological evaluation employing fluorescence microscopy with double-staining acridine orange/ethidium bromide was performed. Acridine orange (AO) easily permeates the cell membrane and results in a green fluorescence of both the nucleus and cytoplasm. Ethidium bromide (EB), however, does not permeate the

Scheme 1. Synthesis of 1,3-thiazine-2,4-diones.

M. Ferreira et al. / European Journal of Medicinal Chemistry 70 (2013) 411e418 Table 1 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

2

82

3

68

4

58

4

77

4

80

4

75

2

78

2

85

413

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 approximately 14% of cells treated with both compounds (Fig. 4).

O NH

1a

O

S

O NH

1b H3CO

O

S

O

1c

O

NH

O

S

O

O NH

1d Cl

S

Cl

O

O NH

1e S

Cl

O

O NH

1f Cl

S

O

O NH

1g

O

OS NH

1h 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

2.2.2. Mitochondrial membrane potential (MMP) measurement and ROS generation Substances which can compromise the integrity of mitochondria 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 (DJm) [37]. This phenomenon can occur due to the release of 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 mitochondrial 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). 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 oxidative 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 production 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 positive 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).

Table 2 CC50 values of compounds against different cancer cell lines. Compound

1a 1b 1c 1d 1e 1f 1g 1h a b

CC50 (mM)a

Selectivity index (SI)b

Vero

L1210

CCRF-CEM

B16F10

MDA-MB-231

Vero/L1210

Vero/CCRF-CEM

Vero/B16F10

Vero/MDA-MB-231

100 100 55 100 60 41 96 100

35 100 63 60 27 30 28 100

e e e 96 49 40 64 100

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 e e 1.0 1.2 1.0 1.5 1.0

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

Data were expressed as mean  SD (n ¼ 3). Defined as the ratio between CC50 of non tumoral cell and tumoral cell.

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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 400). (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 The apoptotic process is mediated by a family of cysteine proteases 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, leukemia 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.

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 phenomenon no longer persists, leading to the conclusion that the decrease in caspase-8 activity consists in a transitory event. 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 mM FCCP as positive control. The decrease of red/green ratio indicates a decrease in mitochondrial membrane potential, and the results are expressed as percentage 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 phosphorylation increasing the ATP production [42]. Mobilizations of the intracellular Ca2þ ([Ca2þ]i) store, for instance depletion or alterations 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 intracellular 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 potential 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

3. Conclusion

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 acquired with a PerkineElmer FT-IR 1600 spectrometer (range 4000e 400 cm1) using KBr for solids samples. 1H NMR (400 MHz) and 13C NMR (100 MHz, fully decoupled) spectra were recorded with a Varian AS-400 spectrometer. Samples were prepared in an appropriate 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].

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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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 temperature. 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 corresponding 1,3-thiazine-2,4-dione 1. 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/cm1: 3445, 3156, 3046, 2855, 1695, 1666, 1630, 1335, 1252, 1187, 1181, 1158. 1H NMR (400 MHz, CDCl3): d 4.07 (s, 2H), 7.34e7.48 (m, 5H), 7.89 (s, 1H), 8.81 (brs, 1H). 13C NMR (100 MHz, CDCl3): d 26.0 (CH2), 123.6 (C), 129.1 (2  CH), 129.5 (2  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. 4.1.2.2. (5Z)-5,6-Dihydro-5-(4-methoxybenzylidene)-1,3-thiazine2,4-dione (1b). White solid, mp 164.0e165.0  C. IR (KBr) nmax/cm1: 3453, 3169, 3063, 2837, 1685, 1600, 1508, 1342, 1304, 1257, 1172. 1H NMR (400 MHz, CDCl3): d 3.85 (s, 3H), 4.10 (s, 2H), 6.97 (d, J ¼ 8.7 Hz, 2H), 7.33 (d, J ¼ 8.7 Hz, 2H), 7.83 (s, 1H), 8.55 (brs, 1H). 13C NMR (100 MHz, CDCl3): d 26.1 (CH2), 55.5 (OCH3), 114.6 (2  CH), 121.1 (C), 126.4 (C), 131.7 (2  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. 4.1.2.3. (5Z)-5,6-Dihydro-5-(3,4-methylenedioxybenzylidene)-1,3thiazine-2,4-dione (1c). White solid, mp 199.0e200.0  C. IR (KBr) nmax/cm1: 3442, 3272, 3053, 2906, 1697, 1657, 1628, 1594, 1497, 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). 13C NMR (100 MHz, DMSO-d6): d 25.3 (CH2), 101.7 (CH2), 108.8 (CH), 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. 4.1.2.4. (5Z)-5-(4-Chlorobenzylidene)-5,6-dihydro-1,3-thiazine-2,4dione (1d). White solid, mp 211.0e213.0  C. IR (KBr) nmax/cm1: 3449, 3165, 3104, 3038, 2844, 1685, 1655, 1602, 1489, 1408, 1328, 1199. 1H NMR (400 MHz, DMSO-d6): d 4.17 (s, 2H), 7.50 (m, 4H), 7.61 (s, 1H), 11.54 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): d 25.1 (CH2), 125.6 (C), 128.9 (2  CH), 131.6 (2  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. 4.1.2.5. (5Z)-5-(2-Chlorobenzylidene)-5,6-dihydro-1,3-thiazine-2,4dione (1e). White solid, mp 156.0e157.0  C. IR (KBr) nmax/cm1: 3455, 3160, 3064, 2853, 1688, 1669, 1620, 1470, 1338, 1188. 1H NMR (400 MHz, DMSO-d6): d 4.06 (s, 2H), 7.40e7.60 (m, 4H), 7.62 (s, 1H), 11.62 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): d 25.2 (CH2), 127.2 (C), 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. 4.1.2.6. (5Z)-5-(2,4-Dichlorobenzylidene)-5,6-dihydro-1,3-thiazine2,4-dione (1f). White solid, mp 176.0e177.5  C. IR (KBr) nmax/cm1: 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 (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 (d, J ¼ 2.0 Hz, 1H), 11.64 (brs, 1H). 13C NMR (100 MHz, DMSO-d6): 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

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-1,3thiazine-2,4-dione (1g). Yellow solid, mp 184.0e186.0  C. IR (KBr) nmax/cm1: 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). 13C NMR (100 MHz, DMSO-d6): d 25.4 (CH2), 125.1 (C), 126.8 (CH), 126.9 (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. 4.1.2.8. (5Z)-5,6-Dihydro-5-[(E)-3-phenyl-2-propenylidene]-1,3thiazine-2,4-dione (1h). Yellow solid, mp 208.0e210.0  C. IR (KBr) nmax/cm1: 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): d 24.6 (CH2), 123.1 (]CH), 123.2 (C), 127.7 (2  CH), 128.9 (2  CH), 129.4 (CH), 136.0 (C), 138.3 (]CH), 142.3 (]CH), 166.6 (C), 168.4 (C). Anal. Calcd. for 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. 4.2. Biological activity 4.2.1. Materials 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 -tetraethylbenzimidazolcarbocyanine 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. 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, supplemented with 1.5 g/L sodium bicarbonate, 10 mM HEPES, pH 7.4, 100 U/mL penicillin G, 100 mg/mL streptomycin and 10% fetal calf serum at 37  C in a humidified atmosphere consisting of 95% air and 5% CO2. Cells were passaged approximately twice a week and cultures with greater than 95% of viable cells in trypan-blue exclusion tests were used for the experiments. 4.2.3. Viability assay The MTT method was used to determine the cell viability [44]. Adherent cells (1  104/well) and suspended cells (1  105/well) were seeded in 96-well plates and incubated for 24 h with increasing concentrations of the compounds, ranging from 15 mM to 100 mM. For the control group, cells were incubated without treatment. After incubation, the old culture medium was replaced with fresh culture medium with 5 mg/mL of MTT, followed by incubation for 1e4 h at 37  C. MTT-formazan crystals were dissolved in 100 mL of DMSO and the absorbance was measured at 540 nm using a micro-well system reader. The CC50 values were calculated through a Hill concentrationeresponse curve. 4.2.4. Morphological evaluation assay A double staining method, with ethidium bromide (3,8diamino-5-ethyl-6-phenylphenanthridinium bromide) and acridine orange (3,6-dimethylaminoacridine), was used to evaluate the morphological alterations and identify the cell death type (necrosis

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versus apoptosis). The cells (1  106/well) were seeded in 12-well plates and incubated with the CC50 concentrations of each compound 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, cells were resuspended in 500 mL of PBS containing 2 mg of ethidium bromide and 0.6 mg of acridine orange. After 2 min, cells were centrifuged again, resuspended in 500 mL of pure PBS and examined under a fluorescence microscope (Nikon, Eclipse TS100). 4.2.5. Flow cytometry analysis To analyze the cell cycle of cells treated with 1,3-thiazine-2,4diones 1, flow cytometry was used following a previously described method [45]. Briefly, the cells (1  106/well) were incubated 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 was discarded and cells were fixed with 200 mL of 70% ethanol for 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 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  106/well) were treated with the CC50 concentration of 1,3-thiazine-2,4diones 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 binding buffer (Millipore) and incubated with 2.5 mL Annexin-VFITC and 5 mL of 20 mg/mL propidium iodide solution for 15 min. 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). 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, fluorescent probe JC-1 was used. The cells (1 106/ 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 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, 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  106 cells/well) and treated for 4 h with 35 mM and 42 mM of 1a and 28 mM and 33.6 mM of 1g. As positive controls, cells were treated with H2O2 for 1 h. At the end of the incubation time, cells were stained with 10 mM DCFH-DA for 30 min at 37  C, washed four times with 1 mL of PBS and resuspended in 500 mL of PBS. The evaluation was performed by quantification of the green fluorescence 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

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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  106 cells/well) and treated with CC50 concentrations of 1a and 1g for 4 and 12 h. The cells were washed with PBS and lysed in 100 mL of lysis buffer containing 50 mM HEPES pH 7.4, 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS), 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/mL pepstatin A, 1 mg/mL leupeptin, and 5 mg/mL aprotinin, homogeneized in a vortex and 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 corresponding substrate for each caspase (caspase-3: 20 mM AcDEVD-AMC; caspase-8: 100 mM Ac-IEDT-AMC; caspase-9: 100 mM Ac-LEDH-AFC; caspase-12: 50 mM Ac-ATAD-AFC). Cleavage of the fluorogenic substrate was detected spectrofluorimetrically (PerkineElmer 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. 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  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 medium was removed and a solution of 300 mL of HBBS with 0.02% Pluronic F127 and Fluo-3 AM (3 mM) 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 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 emission 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. Acknowledgments The authors wish to thank the Central de Análises (Departamento de Química, UFSC, Florianópolis) for spectroscopic analysis. M.F. and L.S.A. are grateful to CAPES (Coordenação de Aperfeiçoamento 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. Appendix A. Supplementary data 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.

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