New Organochalcogen Multitarget Drug: Synthesis and Antioxidant

Article pubs.acs.org/jmc

New Organochalcogen Multitarget Drug: Synthesis and Antioxidant and Antitumoral Activities of Chalcogenozidovudine Derivatives Diego de Souza,† Douglas O. C. Mariano,† Fernanda Nedel,§ Eduarda Schultze,§ Vinícius F. Campos,§ Fabiana Seixas,§ Rafael S. da Silva,† Taiana S. Munchen,† Vinicius Ilha,† Luciano Dornelles,† Antonio L. Braga,‡ Joaõ B. T. Rocha,† Tiago Collares,§ and Oscar E. D. Rodrigues*,† †

LabSelen-NanoBio - Departamento de Química, Universidade Federal de Santa Maria, 97105-900 Santa Maria, Brazil Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, Brazil § Molecular and Cellular Oncology Group, Graduate Program in Biotechnology, Universidade Federal de Pelotas, 96010-610 Pelotas, Brazil ‡

S Supporting Information *

ABSTRACT: In this article we present the synthesis, characterization, and in vitro biological and biochemical activities of new chalcogenozidovudine derivatives as antioxidant (inhibition of TBARS in brain membranes and thiol peroxidase-like activity) as well as antitumoral agents in bladder carcinoma 5637. A prominent response was obtained for the selected chalcogenonucleosides, showing effective antioxidant and antitumoral activities.

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INTRODUCTION Nucleosides have been subject of intense investigation as a potential source of more effective and less toxic drugs. They are crucial components of nucleotides, which are the building blocks of nucleic acids (DNA and RNA). Nucleotides participate in a variety of metabolic processes, including the regulation of cell signaling and enzymatic activities.1 In this context, a number of strategies have been reported for the development of nucleoside analogues, which could potentially modulate metabolic processes controlled by endogenous nucleosides.2 The antiviral and antitumoral activities of synthetic nucleosides can be highlighted, including the nucleosides decitabine,3 clofarabine,4 and 5-azacytidine,5 which have been recently approved for the treatment of leukemic and myelodysplasic syndromes (Figure 1). 3′-Azido-3′-deoxythymidine (1) (Figure 1), also known as zidovudine or AZT, is one of the most usable synthetic nucleosides in medicine. This compound was originally developed as an anti-neoplasic agent,6 although it was approved by the U.S. Food and Drug Administration in 1987 for the treatment of HIV infections.7,8 Despite the therapeutic effectiveness, the major limitation in the clinical use of zidovudine is associated with its side effects.9 Indeed, this molecule may cause toxicity in the bone marrow,10 liver,11 skeletal muscle,12 and heart.13 The molecular toxicity of this nucleoside is still elusive; however, it involves multiple © XXXX American Chemical Society

Figure 1. Structures of biologically active synthetic nucleosides.

mechanisms, including the generation of reactive oxygen species (ROS).14 On the other hand, organochalcogen compounds have been reported to be effective against free radical species and to exhibit other important biological properties, such as antitumor, anti-inflammatory, anti-infective, and antiviral activities.15 Although the synthesis of some chalcogenothymidine derivatives has already been described,16 protocols allowing the preparation of a full spectrum of chalcogenides, encompassing sulfur, selenium, and tellurium, would still be intriguing, especially ones involving the synthesis of original and biologically active azidothymidine derivatives. Received: October 4, 2014

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DOI: 10.1021/jm5015296 J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

Figure 2. Synthesis and biological evaluations of chalcogenozidovudine derivatives.

Scheme 1. Synthesis of Chalcogenozidovudine Derivativesa

(i) Et3N (1.5 equiv), THF, 0 °C; (ii) MsCl (1.1 equiv), THF, 4 h, 0 °C; (iii) (ArY)2 (0.5 mmol), THF (3 mL), EtOH (1 mL), NaBH4 (1.0 equiv), rt, N2. a

Figure 3. Chalcogenozidovudine derivatives.

The combination of the biologically active nucleoside zidovudine with a variety of organochalcogen moieties in a

and their biological exploitation for in vitro antioxidant and antitumoral activities (Figure 2).

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unique molecule affords a new class of prominent bioactive chalcogenonucleosides. Herein we describe the first synthesis of

RESULTS AND DISCUSSION 1. Synthesis of Chalcogenozidovudine Derivatives. For the preparation of the desired compounds, a synthetic

new DNA building block analogues, chalcogenozidovudines, B


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Figure 4. Inhibition of brain lipid peroxidation (TBARS formation) by chalcogenozidovudine derivatives. Data are expressed as mean ± standard error of the mean (SEM) for three different experiments. * indicates a significant difference from DMSO + Fe (p < 0.05).

particularly ones related to the generation of reactive oxygen species (ROS). On other hand, compounds containing selenium appear to be efficient antioxidant protective systems,17 and therefore, some arylselenonucleosides were selected for in vitro evaluations as potential antioxidants. 2.1. Thiobarbituric Acid Reactive Substance Measurements. The final products of lipid peroxidation, which can be quantified by measuring thiobarbituric acid reactive substance (TBARS), were determined in brain homogenates using the methods of Ohkawa et al.18 and Puntel et al.19 with minor modifications. The compounds were analyzed at two different concentrations (100 and 200 μM). At 200 μM, all of the compounds significantly inhibited the production of malondialdehyde in brain homogenates (p < 0.05; Figure 4). Compound 3g showed the highest antioxidant potency (about 80% inhibition of TBARS production). In addition, compounds 3b and 3h also showed excellent results compared with 1. 2.2. Thiol Peroxidase-like Activity Measurements. The thiol peroxidase-like activities of compounds (i.e., the abilities of the zidovudine derivatives to mimic the native antioxidant glutathione peroxidase (GPx)) were evaluated according to the method of Iwaoka and Tomoda.20 In this methodology, benzenethiol (PhSH) is used as a glutathione alternative (Figure 5). Statistical analysis indicated that compounds 3b and 3h decomposed H2O2 more efficiently than 3a, 3d, 3g, 1, and dimethyl sulfoxide (DMSO) as the control group (p < 0.05).

strategy starting from commercially available zidovudine was developed, as depicted in Scheme 1. First, zidovudine was treated with mesyl chloride in tetrahydrofuran (THF) and Et3N, affording the respective mesylate 2. Furthermore, a variation in the search for an effective protocol to afford the respective chalcogenozidovudines 3 was performed using diphenyl diselenide as standard dichalcogenide moiety. In this context, the most effective protocol was to employ THF/EtOH as the solvent and NaBH4 as a reducing agent at room temperature for 6 h, affording the respective product in 55% yield (Figure 3a). The use of other reducing agents, such as Zn0, did not allow the preparation of compound 3a in a more effective yield. Thereby, the same procedure was extended to other dichalcogenides, and the preparation of a small library of new chalcogenozidovudine derivatives was performed, as depicted in Figure 3. In order to check the effects of the dichalcogenide moiety in the reaction course, different diaryl dichalcogenides were applied for this protocol. Figure 3 shows a small relation of electronic and steric effects in terms of the dichalcogenide moiety. In general, the attachment of activating groups to the aromatic ring gave slightly better yields than attachment of deactivating groups (Figure 3, entries 3b, 3c, and 3e vs 3f and 3i). Thus, the slightly higher yields obtained with compounds with activating groups as substituents on the aromatic rings can probably be explained by the higher nucleophilicities of the chalcogenolate anions with these substituents. In terms of steric effects, the most hindered reagent, dimesityl diselenide, gave the desired compound 3d less effectively compared with the pand o-methyl-substituted diaryl diselenides (3b and 3c, respectively). Comparing the chalcogen atoms, sulfur and subsequently selenium achieved better yields than tellurium (examples 3a, 3j, and 3k, respectively). These results can be rationalized on the basis of the stabilities of the respective chalcogenolate anions under the reaction conditions. The use of a variety of chalcogenium moieties allowed the preparation of a small library of new chalcogenozidovudine derivatives with different electronic and steric properties, which was very convenient to use in the proposed exploration of biological interactions. 2. Antioxidant Studies. A serious drawback to the development and application of drugs to some therapies especially as antitumoral compoundsis their toxicity, which may cause various side effects. Despite its effectiveness as an antiretroviral and antitumoral drug, some side effects related to the continued use of zidovudine have been observed,

Figure 5. GPx-like activity of chalcogenozidovudine derivatives. All of the compounds were tested at 500 μM (n = 3 independent experiments done in triplicate). * indicates a significant difference from the control group (p < 0.05). C


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nucleoside are unknown, but hypersensitivity may play a role. On the basis of this and the results obtained previously, compounds 3a, 3b, and 3h were selected along with 1 in order to analyze the mitochondrial metabolic function. The hepatic mitochondrial metabolic function was assessed by the conversion of MTT to a dark-violet formazan product by mitochondrial dehydrogenases.26 The MTT reduction assay showed that the chalcogenozidovudine derivatives 3a, 3b, and 3h did not modify the cell viability compared to the control (DMSO) slices group (p > 0.10; Figure 7). Compound 1 did not interfere with cell viability (p > 0.10) compared to the control group, but nevertheless, at concentrations of 200, 100, and 50 μM it caused a significant reduction in cell viability compared with compound 3h at 200 μM. In fact, compound 3h exhibited a concentration-dependent tendency to increase the reduction of MTT. A weak but similar tendency was also observed for the highest concentrations of compounds 3a and 3b. Furthermore, to verify the presence of more specific hepatotoxic effects of 1 and chalcogenonucleosides derivatives 3a, 3b, and 3h, we analyzed the activity of serum aminotransferases (AST and ALT). Increases in the serum ALT and AST activities can result from either nonspecific cell damage or from more subtle changes in membrane permeability, which can indicate severe to slight liver damage. However, there was no significant change in AST and ALT release from slices incubated with 1, 3a, 3b, or 3h compared to the control. 4. Antitumoral Studies. Since 1965, great interest has been attached to the possible role of free radicals in cancer tissues.27 Most researchers have agreed that oxygen radicals participate in both initiation and promotion of cancer. At the initiation stage, oxygen radicals in connection with various carcinogens could change normal cellular genetic material to neoplasic genetic composition. At the promotion stage, the participation of free radicals was first suggested on the grounds of the effects of organic peroxides, which promoted cancer development.28 Inexorably, antitumoral−antioxidant compounds can be described as chameleonic drugs because of the possibility of combining cytotoxic properties against cancer cells and chemoprotection against ROS, which are normally found at high levels in neoplasic tissues.29 On the basis of this and the results presented above, compounds 3a, 3b, and 3h, which

The times required to oxidize 50% of the benzenethiol (i.e., t50) during the reduction of hydrogen peroxide are depicted in Figure 6. In line with the data presented in Figure 6, the t50 values for compounds 3h and 3b were 4−5 times lower than those for DMSO and 1 (Figures 5 and 6).

Figure 6. Time required to oxidize 50% of PhSH (t50). All of the compounds were tested at μM. Data are expressed as mean ± SEM of the GPx-like activities determined in three different experiments.

3. Hepatotoxicity. Drug hepatotoxicity is the leading cause of acute liver failure. While the overall incidence of druginduced liver injury is infrequent,21 the impact is significant. At a regulatory level, hepatotoxicity is the main reason for postmarketing regulatory decisions including drug withdrawal.22 Chronic zidovudine therapy is a well-established case of clinically apparent liver injury. The continued use of this compound is associated with modest serum enzyme elevations that are generally transient and asymptomatic and do not require dose modification, as the liver injury is usually mild to moderate and self-limiting.23 Although serum enzymes are only modestly elevated and may be initially normal, jaundice and signs of liver dysfunction can eventually be associated with acute hepatic failure and death.24 The hepatic failure may be due to inhibition of the mitochondrial γ-polymerase by zidovudine, leading to mitochondrial dysfunction.25 The causes of other forms of clinically apparent liver injury due to this

Figure 7. Effects of the treatment with 1 and chalcogenozidovudine derivatives 3a, 3b, and 3h on the reduction of MTT by slice from adult rats. Data are expressed as mean ± standard error of the mean (SEM) for three different experiments. Significance was assessed by one-way analysis of variance (ANOVA) followed by the Newman−Keuls multiple comparisons test. Columns that do not share the same superscript letter were different at p < 0.05. D


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Figure 8. Inhibitory effects of 1, 3a, 3b, and 3h on the growth of 5637 cells after 24, 48, and 72 h of incubation. Data are expressed as mean ± SEM, and differences are considered significant for p < 0.05. Upper-case letters indicate differences between treatment time and lower-case letters indicate differences between concentrations.

exhibited prominent antioxidant properties, were selected for screening as in vitro antitumoral agents. 4.1. Determination of Synthetic Nucleoside Cytotoxicity against Bladder Carcinoma Cells (5637). Compound 1 and its analogues 3a, 3b, and 3h showed in vitro cytotoxicity against human bladder carcinoma cells (5637) after 24, 48, or 72 h of exposure. It is interesting to note that the maximal cytotoxicity was obtained after 48 h of exposure to 1, 3b or 3h at 50 μM. After 48 h of incubation, 1, 3b, and 3h inhibited cell growth by 50% (Figure 8). In addition, 1 inhibited cell growth more than 50% at all of the tested concentrations. Previous studies have demonstrated tumor cell growth inhibition by zidovudine at low concentrations as demonstrated here.30 The analogue 3a inhibited cell growth more than 50% only at higher concentrations (100 and 200 μM). In addition, no effect of the analogue exposure time was observed in cell growth inhibition. The IC50 of each treatment was calculated using a nonlinear best-fit line with the growth inhibition rate (%) after 48 h of incubation. These results showed that after 48 h, 1 was more effective in inhibiting tumor growth than its derivatives (Figure 8 and Table 1) at lower concentrations. However, at higher concentrations the chalcogenozidovudine derivatives 3a, 3b, and 3h) were more effective in inhibiting tumor growth than 1 (Figure 8). Furthermore, the chalcogenonucleosides derivatives did not cause cellular injury even at the highest concentrations tested (Figure 7). 4.2. Apoptosis Analysis. The annexin V-PE/7AAD staining assay was performed to determine whether compound 1 and chalcogenozidovudine derivatives 3a, 3b, and 3h could induce apoptosis in 5637 cells. Annexin V binds to cells that expose phosphatidylserine on the outer layer of the cell membrane, a

Table 1. IC50 Values for Zidovudine (1) and Chalcogenozidovudine Derivatives against Human Bladder Cancer Cellsa group

IC50 (μM)

1 3a 3b 3h

7 ± 1.421 78.85 ± 2.481 51.63 ± 7.861 40.4 ± 3.384

a

The 5637 cells were treated with different concentrations of 1, 3a, 3b, and 3h and incubated for 48 h. Cell viability was determined by the MTT assay. All data were obtained from three independent experiments and are presented as mean ± SEM.

characteristic feature of cells entering apoptosis. The results (Figure 9) indicate that cells treated with 1 had a similar percentage of apoptosis as found in control cells. However, exposure of 5637 cells to 3a, 3b, or 3h (100 μM) increased the percentage of annexin V-PE and 7-ADD staining (early and late apoptosis) to 90.74%, 89.60%, and 92.80%, respectively. In addition, compound 3h induced a high percentage of apoptosis (71.12%) at 50 μM compared with 1 (7.74%) and control (7.36%). These results indicate the pro-apoptotic properties of compounds 3a, 3b, and 3h in 5637 cells. 4.3. DNA Fragmentation Analysis. Because these compounds may interfere with DNA synthesis, we evaluated the induction of apoptosis-associated DNA fragmentation using TdT-mediated dUTP nick end (TUNEL) staining. Treatments with 3a, 3b, and 3h significantly increased the number of TUNEL-positive cells compared with the control and 1 (Figure 10A). E


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4.4. Live/Dead Assay. To better understand the cytotoxic properties of the compounds, a two-color fluorescence cell viability assay was conducted following the manufacturer’s instructions. Figure 10B−D shows representative images of this assay. The treatment with 3h at 100 μM (D) showed many more dead cells than 1 (C) and control (B). 4.5. Gene Expression. We investigated the gene expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax. Bcl-2 mRNA levels were significantly lower in cells exposed to compounds 1, 3a, 3b, and 3h at 100 μM compared with the levels in control cells (Figure 11B). Moreover, Bax mRNA levels were significantly higher in these same groups, except in the case of 1, for which the Bax mRNA levels did not differ from those of the controls (Figure 11A). In addition, a predominance of Bax to Bcl-2 for compounds 3a and 3b was observed at a concentration of 100 μM compared with the controls, where Bcl-2 was predominant to Bax, showing that the Bax:Bcl-2 ratio is affected by these compounds (Figure 11E). We also investigated the mRNA expression for the caspase-9 and survivin genes. Compounds 3a and 3b at 100 μM induced significant increases in caspase-9 mRNA levels in comparison with the controls (Figure 11C), while compounds 3a, 3b, and

Figure 9. Flow cytometry analysis of annexin V-PE/7AAD staining of 5637 cells treated with 1, 3a, 3b, or 3h at 50 or 100 μM for 48 h. Data are expressed as mean ± SEM. Early apoptosis represents cells that were annexin V-PE(+)/7AAD(−), and late apoptosis/dead cells represent cells that were annexin V-PE(+)/7AAD(+). * indicates significant differences between the late apoptosis/dead cells rate relative to the control, and # indicates significant differences between the early apoptosis rate relative to the control.

Figure 10. Apoptosis-associated DNA damage and cytotoxicity induced by chalcogenozidovudine derivatives. (A) Data from the TUNEL staining assay. TUNEL-positive cells represent cells with DNA fragmentation. Data are expressed as mean ± SEM. Different letters represent significant differences. (B−D) Data from the live/dead assay. Shown are representative images of (B) the control, (C) 1, and (D) 3h (100 μM). Live cells are shown in green and dead cells in red. The treatment with 3h showed the highest rate of dead cells. Scale bars indicate 100 μm. F


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Figure 11. Effects of 1, 3a, 3b, and 3h on apoptotic-related gene expression. Data represent mean ± SEM of experiments performed in triplicate. Different letters indicate significant differences among means.

of Bcl-2 was downregulated by compounds 3a, 3b, and 3h, whereas Bax expression was upregulated. Caspases play a central role in apoptosis, a well-studied pathway of programmed cell death. The activation of caspase-9 is believed to be a well-defined outcome of the release of mitochondrial cytochrome c into the cytoplasm and its subsequent association with the Apaf-1 protein. The assembly of cytochrome c, Apaf-1, and procaspase-9, called apoptosome, triggers the activation of caspase-9.38 Here, the caspase-9 mRNA levels were significantly increased by the treatment with compounds 3a, 3b, and 3h, showing that caspase-9 is involved in mediating the apoptotic effects associated with these compounds. In 5637 cells, compounds 3a, 3b, and 3h also modulate the expression of the anti-apoptotic gene survivin,39 which was substantially downregulated at the mRNA level when tumor cells were treated with the compounds. The gene expression

3h induced significant decreases in survivin gene expression in comparison with the controls (Figure 11D). Many genes are involved in the regulation of apoptosis. Bcl-2 is an anti-apoptotic regulator, while Bax is a pro-apoptotic regulator.31 The balance between Bcl-2 and Bax expression plays an important role in sustaining cell morphology and function. Several studies have shown that Bcl-2 overexpression may disrupt the regulation of the pro-apoptotic proteins Bax and Bak.32 Moreover, an increase in Bcl-2 expression prevents the release of cytochrome c from the mitochondria, thereby inhibiting the activation of caspases, such as caspase-9 and caspase-3, and preventing apoptosis.33,34 Other studies have demonstrated that Bcl-2 may act as a mitochondrial membrane channel protein.35 Thus, cells are active when Bcl-2 is overexpressed and are apoptotic (die) if Bax is hyperexpressed.36,37 In the present study, we found that the expression G


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5′-(Phenylseleno)zidovudine (3a). 1H NMR (CDCl3, 400 MHz): δ 9.27 (s, 1H), 7.58−7.51 (m, 2H), 7.32−7.26 (m, 4H), 6.12 (t, J = 6.55 Hz, 1H), 4.25−4.14 (m, 2H), 3.24 (d, J = 5.1 Hz, 2H), 2.53−2.30 (m, 2H), 1.86 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.8, 149.9, 135.4, 132.3, 129.5, 127.9, 111.4, 84.9, 82.8, 62.8, 37.4, 29.7, 12.4. HRMS: calcd for C16H18N5O3Se [M + H]+ 408.0575, found 408.0575. Physical state: yellow oil. Yield: 55%. Purity: 98.8%. 5′-(4-Methylphenylseleno)zidovudine (3b). 1H NMR (CDCl3, 400 MHz): δ 8.55 (s, 1H), 7.44 (d, J = 8.08 Hz, 2H), 7.28 (s, 1H), 7.11 (d, J = 7.85 Hz, 2H), 6.12 (t, J = 6.53 Hz, 1H), 4.21−4.16 (m, 1H), 4.10− 4.05 (m, 1H), 3.20 (d, J = 5.08 Hz, 2H), 2.49−2.43 (m, 2H), 2.35 (s, 3H), 1.90 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.4, 149.9, 137.9, 135.4, 132.8, 130.5, 125.1, 111.5, 84.7, 83.0, 62.9, 37.5, 30.4, 21.1, 12.7. HRMS: calcd for C17H20N5O3Se [M + H]+ 422.0731, found 422.0723. Physical state: yellow oil. Yield: 58%. Purity: 96.2%. 5′-(2-Methylphenylseleno)zidovudine (3c). 1H NMR (CDCl3, 400 MHz): δ 9.64 (s, 1H), 7.49 (d, J = 7.70 Hz, 1H), 7.23−7.09 (m, 4H), 6.12 (t, J = 6.50 Hz, 1H), 4.25−4.18 (m, 1H), 4.15−4.08 (m, 1H), 3.27−3.20 (m, 2H), 2.46 (s, 3H), 2.41−2.32 (m, 1H), 2.06−1.92 (m, 1H), 1.85 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ 164.0, 150.3, 139.1, 135.7, 131.5, 130.3, 127.4, 126.9, 111.2, 84.9, 82.7, 62.9, 37.5, 28.7, 22.5, 12.2. HRMS: calcd for C17H20N5O3Se [M + H]+ 422.0731, found 422.0732. Physical state: yellow oil. Yield: 60%. 5′-(2,4,6-Trimethylphenylseleno)zidovudine (3d). 1H NMR (CDCl3, 400 MHz): δ 8.75 (s, 1H), 7.18 (s, 1H), 6.95 (s, 2H), 6.18 (t, J = 6.6 Hz, 1H), 4.34−4.13 (m, 1H), 4.13−4.02 (m, 1H), 3.70 (d, J = 5.09 Hz, 2H), 2.99 (s, 9H), 2.48−2.02 (m, 2H), 1.94 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.5, 150.0, 142.3, 140.0, 135.4, 128.8, 126.2, 111.5, 84.9, 82.6, 62.8, 37.5, 29.7, 24.4, 21.9, 12.6. HRMS: calcd for C19H24N5O3Se [M + H]+ 450.1044, found 450.1045. Physical state: yellow oil. Yield: 45%. Purity: 97.8%. 5′-(4-Methoxyphenylseleno)zidovudine (3e). 1H NMR (CDCl3, 400 MHz): δ 8.70 (s, 1H), 7.75 (s, 1H), 7.53 (d, J = 7.78 Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 5.72 (t, J = 6.5 Hz, 1H), 4.40−4.25 (m, 1H), 4.20−4.08 (m, 1H), 3.84 (s, 3H), 3.29−3.26 (m, 2H), 2.60−2.28 (m, 2H), 1.94 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.5, 159.7, 149.9, 135.5, 132.1, 118.7, 115.2, 111.3, 84.9, 83.0, 62.9, 55.3, 37.4, 29.7, 12.6. HRMS: calcd for C17H20N5O4Se [M + H]+ 438.0681, found 438.0683. Physical state: dark-yellow oil. Yield: 60%. 5′-(3-Trifluoromethylphenylseleno)zidovudine (3f). 1H NMR (CDCl3, 400 MHz): δ 9.15 (s, 1H), 7.79 (s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.40 (t, J = 5.8 Hz, 1H), 7.26 (s, 1H), 6.07 (t, J = 6.5 Hz, 1H), 4.23−4.17 (m, 1H), 4.09−4.04 (m, 1H), 3.40−3.23 (m, 2H), 2.50−2.35 (m, 2H), 1.84 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.7, 150.6, 135.8, 135.1, 133.5, 131.7 (q, J = 32.6 Hz), 130.4, 129.6, 128.8, 128.1, 126.7, 110.9, 85.4, 82.5, 62.2, 37.2, 29.7, 12.6. HRMS: calcd for C17H16F3N5NaO3Se [M + Na]+ 498.0268, found 498.0258. Physical state: dark-yellow oil. Yield: 42%. 5′-(1-Naphthylseleno)zidovudine (3g). 1H NMR (CDCl3, 400 MHz): δ 8.85 (s, 1H), 8.40 (d, J = 8.29 Hz, 1H), 7.91−7.83 (m, 3H), 7.63−7.52 (m, 2H), 7.49−7.42 (m, 1H), 7.23 (s, 1H), 6.07 (t, J = 6.53 Hz, 1H), 4.19−4.09 (m, 2H), 3.33−3.23 (m, 2H), 2.48−2.32 (m, 2H), 1.86 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.4, 149.9, 135.4, 132.8, 129.1, 128.9, 127.2, 126.9, 126.4, 125.8, 111.2, 85.5, 83.0, 63.1, 37.3, 30.2, 12.2. HRMS: calcd for C20H20N5O3Se [M + H]+ 458.0731, found 458.0734. Physical state: yellow oil. Yield: 45%. Purity: 95.8%. 5′-(4-Chlorophenylseleno)zidovudine (3h). 1H NMR (CDCl3, 400 MHz): δ 9.85 (s, 1H), 7.49−7.45 (m, 2H), 7.28−7.22 (m, 2H), 7.20 (s, 1H), 6.08 (t, J = 6.43 Hz, 1H), 4.22−4.12 (m, 1H), 4.11−4.01 (m, 1H), 3.28−3.18 (m, 2H), 2.49−2.36 (m, 2H), 1.86 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 164.2, 150.3, 135.7, 133.9, 129.7, 127.2, 111.1, 85.2, 82.7, 62.7, 37.4, 30.0, 12.4. HRMS: calcd for C16H17ClN5O3Se [M + H]+ 442.0185, found 442.0173. Physical state: yellow oil. Yield: 67%. Purity: 97.5%. 5′-(((4-Methylphenyl)carbonyl)seleno)zidovudine (3i). 1H NMR (CDCl3, 400 MHz): δ 9.90 (s, 1H), 8.0 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.24 (s, 1H), 6.19 (t, J = 6.44 Hz, 1H), 4.61−4.45 (m, 2H), 4.42−4.28 (m, 1H), 4.26−4.15 (m, 1H), 2.62−2.26 (m, 5H), 1.69 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 168.2, 162.5, 149.2, 144.5, 134.6, 130.5, 129.8, 129.5, 129.4, 128.1, 110.9, 85.6, 82.1, 62.8,

data confirm the results of flow cytometry that compounds 3a, 3b, and 3h are more efficient pro-apoptotic agents than 1.

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CONCLUSION Here we have proposed the synthesis of new nucleosides derived from zidovudine containing Se, Te, or S. The methodology used allowed the preparation of a small library of chalcogenozidovudine derivatives in good yields and in a modular fashion. The synthesized compounds were evaluated for biological applications as antioxidant and antitumoral agents against bladder carcinoma 5637 cells and showed effectiveness in all of the bioactivities tested here. Studies performed to evaluate the toxicities of the new chalcogenozidovudines showed a lower value in this parameter compared with commercial zidovudine, even at higher concentrations. In this context, very convenient bioapproaches were observed for these new chalcogenonucleosides, which showed different and desired bioresponses for new drug candidates. Additional tests against other carcinomas and new generations of chalcogenozidovudine derivatives are being developed in our laboratory.

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EXPERIMENTAL SECTION

Chemistry. All of the reactions were run under an atmosphere of dry nitrogen or argon unless otherwise noted. DMSO, thiobarbituric acid (TBA), and malondialdehyde were obtained from Sigma (St. Louis, MO). All of the other chemicals were of analytical grade and obtained from standard commercial suppliers. The NMR spectra were recorded with a Bruker DPX-400 spectrometer with chemical shifts (δ) expressed in parts per million (in CDCl3 with Me4Si as an internal standard). Data are reported as follows: chemical shift (multiplicity, coupling constant(s), number of protons). Multiplicities are denoted as follows: s = singlet, d = doublet, t = triplet, q = quartet, br s = broad singlet, m = multiplet. All of the test compounds showed >95% purity as determined by LC−MS. LC−MS analyses used the following conditions: Shimadzu LC-20AD pump, Shimadzu SIL-20A autosampler, Shimadzu CTO-20A column oven (40 °C), Shimadzu SPDM20A DAD detector. Column: Phenomenex ref 00F-4252-E0, Luna C18(2)5u, 5 μm, 100 Å, 4.6 mm × 150 mm. Gradient: 20−100% acetonitrile in water containing 0.01% formic acid. Flow rate: 0.7 mL/ min. MS: Bruker MicrOTOF QII with an electrospray ionization (ESI) source. 5′-O-(Mesyl)zidovudine (2). To a two-neck round-bottom flask under an argon atmosphere was added 1 mmol of zidovudine (1) in 7 mL of THF. After dissolution of the zidovudine, the system was cooled to 0 °C, and triethylamine (1.5 mmol) was added. After 10 min, still at 0 °C, mesyl chloride (1.1 mmol) diluted in 3 mL of THF was added dropwise. Then the ice bath was removed, and the system was allowed to react for 2 h at room temperature. After this period, the reaction mixture was extracted with a saturated solution of NH4Cl (∼20 mL), and the organic phase was extracted with dichloromethane (3 × 20 mL). The organic phases were combined and dried over MgSO4, and the solvent was evaporated under reduced pressure. The product was crystallized in ethyl acetate and dried using a high-vacuum pump. The yield of mesylate 2 was 92%. Representative Procedure for the Preparation of Compounds 3a−k. To a two-neck round-bottom flask under an argon atmosphere were added diaryl dichalcogenide (0.6 mmol), THF (3 mL), and ethanol (2 mL). Then NaBH4 (1,0 equiv) was added, and the reaction mixture was stirred until the color disappeared. Subsequently, mesylate 2 (1 mmol) dissolved in THF (3 mL) was added dropwise to the reaction flask. The system was stirred at room temperature for 6 h. After the completion of the reaction, the mixture was quenched with a saturated solution of NH4Cl and extracted with CH2Cl2. The crude product was purified on a chromatographic column employing a 90:10 mixture of dichloromethane and ethanol as the solvent. Compounds 3a−k were obtained in yields of 40−83%. H


Article


final concentration of 200, 100, or 50 μM. The slices were incubated for 2 h at 37 °C. After this period of exposure, 10 μL of a solution of 5 mg/mL MTT was added and allowed to react for 30 min at 37 °C. Then the solution was removed and 500 μL of DMSO/well was added to dissolve the crystalline formazan product formed inside the liver slices. The absorbance at 570 and 700 nm was read spectrophotometrically using a microplate reader. The results were expressed as a percentage of the absorbance of nontreated cells. Cell Culture. Human bladder carcinoma cells (5637) were obtained from Rio de Janeiro Cell Bank (PABCAM, Federal University of Rio de Janeiro, RJ, Brazil). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/ streptomycin, purchased respectively from Vitrocell Embriolife (Campinas, Brazil) and Gibco (Grand Island, NY, USA). Cells were grown at 37 °C in an atmosphere of 95% humidified air and 5% CO2. The experiments were performed with cells in the logarithmic phase of growth. Determination of Cytotoxicity. The viability of 5637 cells was determined by measuring the reduction of soluble MTT to water insoluble formazan.40 Briefly, cells were seeded at a density of 2 × 104 cells per well in a volume of 100 μL in 96-well plates and grown at 37 °C in a 5% CO2 atmosphere for 24 h before use in the cell viability assay. Cells were then incubated with different concentrations of zidovudine, 3a, 3b, or 3h (200−12.5 μM) for 24, 48, or 72 h. These components were previously dissolved in DMSO and added to the medium supplemented with 10% FBS to the desired concentrations. The final DMSO concentration in the medium never exceeded 0.25%, and an additional group was exposed to an equivalent concentration of this solvent. After the incubation period, the medium was removed, and subsequently 180 μL of medium and 20 μL of MTT (5 mg MTT/ mL solution) were added to each well. The plates were incubated for an additional 3 h, and the medium was discarded. A 200 μL aliquot of DMSO was added to each well, and the formazan was solubilized on a shaker for 5 min at 150 rpm. The absorbance of each well was read on a microplate reader (MARCA) at a test wavelength of 492 nm. Cell inhibitory growth was determined as follows: inhibitory growth = [1 − (Abs492 for treated cells)/(Abs492 for control cells)] × 100%. All of the observations were validated using at least two independent experiments in triplicate for each experiment. Live/Dead Assay. Cells were cultured in 96-well culture plates at a density of 2 × 104 cells per well and grown at 37 °C in a 5% CO2 atmosphere for 24 h. Cells were then incubated with 100 or 50 μM zidovudine, 3a, 3b, or 3h for 48 h. At the end of the incubation period, the cells were washed and stained using the LIVE/DEAD Viability/ Cytotoxicity Assay Kit (Invitrogen). Live cells were able to take up calcein and could be analyzed by the green fluorescence emission (488 nm). Ethidium bromide homodimer could diffuse through the nowpermeable membrane of dead cells and bind to DNA, which was detected by the red fluorescence signal (546 nm). The live/dead assay was analyzed with an Olympus IX71 fluorescence microscope (Olympus Optical Co., Tokyo, Japan) by multicolor imaging. The recorded images were analyzed using Cell software (Cell, Olympus, Tokyo, Japan). Apoptotic Assay. The Guava Nexin assay (Guava Technologies) was carried out following the manufacturer’s instructions. Cells were treated with 100 or 50 μM zidovudine, 3a, 3b, or 3h for 48 h. Briefly, 2.0 × 104 to 1.0 × 105 cells (100 μL) were added to 100 μL of Guava Nexin reagent. Cells were incubated in the dark at room temperature for 20 min, and samples (2.000 cells per well) were then acquired on the Guava EasyCyte flow cytometry system. In this assay, annexin Vnegative and 7-AAD-positive indicates nuclear debris, annexin Vpositive and 7-AAD-positive indicates late apoptotic cells, annexin Vnegative and 7-AAD-negative indicates live healthy cells, and annexin V-positive and 7-AAD-negative indicates early apoptotic cells. DNA Fragmentation Analysis. The Guava TUNEL assay (Guava Technologies) was conducted following the manufacturer’s instructions. Cells treated with zidovudine, 3a, 3b, or 3h for 48 h were collected, fixed, and stained with DNA Labeling Mix (Merck KGaA,

38.1, 21.9, 21.3, 12.3. HRMS: calcd for C18H19N5NaO4Se [M + Na]+ 472.0500, found 472.0500. Physical state: light-yellow oil Yield: 47%. 5′-(Phenylthio)zidovudine (3j). 1H NMR (CDCl3, 400 MHz): δ 9.51 (s, 1H), 7.42 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.29− 7.22 (m, 2H), 6.11 (t, J = 6.5 Hz, 1H), 4.29−4.20 (m, 1H), 4.12−4.05 (m, 1H), 3.32 (d, J = 5.1 Hz, 2H), 2.49−2.33 (m, 2H), 1.84 (s, 3H). 13 C NMR (CDCl3, 100 MHz): δ = 164.0, 150.3, 135.6, 134.9, 129.3, 127.0, 111.2, 85.2, 82.5, 62.2, 37.4, 36.3, 12.3. HRMS: calcd for C16H17N5NaO3S [M + Na]+ 382.0950, found 382.0942. Physical state: light-yellow oil. Yield: 83%. 5′-(Phenyltelluro)zidovudine (3k). 1H NMR (CDCl3, 400 MHz): δ 9.22 (s, 1H), 7.87−7.70 (m, 1H), 7.45−7.19 (m, 5H), 6.22 (t, J = 6.61 Hz, 1H), 4.42−4.27 (m, 1H), 4.25−4.12 (m, 1H), 3.95−3.79 (m, 2H), 2.52−2.30 (m, 2H), 1.98 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ = 163.4, 149.9, 135.4, 132.8, 129.1, 127.2, 125.8, 111.2, 85.5, 83.0, 63.1, 37.3, 30.2, 12.2. HRMS: calcd for C16H18N5O3Te [M + H]+ 458.0472, found 458.0473. Physical state: reddish oil. Yield: 40%. LC−MS Analysis of Compounds 3a, 3b, 3d, 3g, and 3h. HPLC: Shimadzu LC-20AD pump, Shimadzu SIL-20A autosampler, Shimadzu CTO-20A column oven (40 °C), Shimadzu SPD-M20A DAD Detector. Column: Phenomenex ref 00F-4252-E0, Luna C18(2)5u, 5 μm, 100 Å, 4.6 mm × 150 mm. Gradient: 20−100% acetonitrile in water containing 0.01% formic acid. Flow rate: 0.7 mL/ min. MS: MicrOTOF QII Bruker, with ESI-source. HPLC Analysis for Zidovudine. The purity of zidovudine (>99%) was determined using the protocol described by dos Santos et al.43 HPLC: Shimadzu LC-20AT pump, Shimadzu DGU degasser, Shimadzu CBM-20A controller, Shimadzu SPD-20A UV/vis detector. Column: VertiSep GES C18 HPLC column (150 mm × 4.6 mm, 5 μm, standard female fittings). Gradient: starting with 20:80 (v/v) methanol/water, a linear gradient was applied for 5 min up to 50:50 (v/v) methanol/water, after which elution with 20:80 (v/v) methanol/ water was used. Flow rate: 1.0 mL min−1. Animals. Adult male Wistar rats (250−350 g) from our own breeding colony (Animal Householding, UFSM, Brazil) were maintained in a temperature-controlled room (22−25 °C) on a 12 h light/dark cycle with water and food ad libitum. Animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources of this university. Tissue Preparation. Animals were killed by decapitation. Brains were removed and homogenized in 10 mM Tris-HCl buffer (1:10 w/v, pH 7.4) and centrifuged at 2000 rpm for 10 min. The low-speed supernatant fraction obtained (S1) was maintained in ice-cold water for the TBARS assay. TBARS Assay. The end products of the lipid peroxidation, through the measurement of TBARS, were determined in tissue samples by the methods of Ohkawa et al.18 and Puntel et al.19 with minor modifications. Aliquots of the homogenate were pipetted and incubated in a medium containing distilled water and 10 mM TrisHCl buffer (pH 7.4) in the absence (DMSO - vehicle) or presence of compouns (at a final concentration of 100 or 200 μM) and 60 μM iron(II) sulfate (FeSO4). Only tissue, buffer, and water were added to the blank group. Diphenyl diselenide was used as a positive control. The amount of TBARS produced was measured at 532 nm using MDA as an external standard. Thiol Peroxidase-like Activity Assay. The catalytic activities of compounds such as a GPx-like model enzyme were evaluated according to the method of Iwaoka and Tomoda.20 using benzenethiol (PhSH) as a glutathione alternative. PhSH (15 mM) and H2O2 (20 mM) were diluted in methanol. Compounds or DMSO (vehicle) were pipetted into a quartz cuvette containing methanol with or without 500 μM chalcogenozidovudine derivative. After this, PhSH and H2O2 were pipetted. The oxidation of PhSH was monitored at 305 nm for 180 s. Cell Viability Assay. The reduction levels of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were determined as an index of the dehydrogenase enzymes functions, which are involved in the cellular viability. Slices obtained from livers of healthy rats were added to a medium containing Krebs Ringer buffer (pH 7.4) for 10 min. Then 10 μL of zidovudine, 3a, 3b, or 3h was added to a I



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Darmstadt, Germany) and Anti-BrdU Staining Mix. Cells were incubated in the dark at room temperature for 30 min, and samples were acquired on flow cytometry (Guava EasyCyte flow cytometry system). In this assay, terminal deoxynucleotidyl transferase (TdT) catalyzes the incorporation of 5-bromo-2′-deoxyuridine (BrdU) residues into the fragmenting nuclear DNA of apoptotic cells at the 3a-hydroxyl ends by nicked-end labeling. Tetramethylrhodamineconjugated anti-BrdU antibody binds to the incorporated BrdU residues, labeling the mid- to late-stage apoptotic cells. Real-Time PCR Gene Expression. For real-time polymerase chain reaction (PCR), cells were seeded in six-well flat bottom plates at a density of 2 × 105 cells per well and grown at 37 °C in a 5% CO2 atmosphere for 24 h. Cells were then incubated with 100 or 50 μM of zidovudine, 3a, 3b, or 3h for 48 h. After this period, cells were washed with phosphate-buffered saline (Gibco), and RNA extraction was performed. Total RNA extraction, cDNA synthesis, and real-time PCR were conducted as previously described.41,42 Briefly, RNA samples were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and samples were DNase-treated with a DNA-free kit (Ambion) following the manufacturer’s protocol. First-strand cDNA synthesis was performed with 2 μg of RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s protocol. Real-time PCRs were run on a Stratagene Mx3005P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems) and the appropriate primers. Statistical Analysis. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test when appropriate. All values are presented as mean ± SEM, and the differences were considered significant when p < 0.05 and p < 0.01. Data sets from MTT and quantitative real-time PCR were analyzed using two-way ANOVA followed by a Tukey test for multiple comparisons. Two factors were considered: used compound (four levels) and compound concentration (two levels). Significance was considered at p < 0.05 in all analyses. Data from the annexin V-PE/ 7AAD assay were analyzed by the χ2 test.

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ASSOCIATED CONTENT

NMR spectra, mass spectra, LC−MS data, and a full description of the biological activities. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +55 55 3220 8761. E-mail: [email protected]. br. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge CAPES, CNPq (Ed. Universal 478054/2012-2, INCT NanoBiosimes), and FAPERGS (Ed. PRONEM 11/2080-9) for financial support. We also thank CEBIME-UFSC for the LC−MS analysis and HUSM-UFSM.

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REFERENCES

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S Supporting Information *

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ABBREVIATIONS

Apaf-1, apoptosis protease-activating factor-1; Bak, proapoptotic members; BAX, pro-apoptotic members; Blc-2, anti-apoptotic proteins; HRMS, high-resolution mass spectrometry; MsCl, methanesulfonyl chloride; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide J


Article

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