Synthesis and Biological Evaluation of Novel 1,3-Benzoxathiol-2-one Sulfonamides against Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snakes

This work describes the synthesis of new 1,3-benzoxathiol-2-one sulfonamides and evaluation of their ability to inhibit some in vitro (coagulant, proteolytic and hemolytic) and in vivo (hemorrhagic, edematogenic and lethality) toxic activities of Bothrops jararaca and B. jararacussu venoms. Compounds have been synthesized from the coupling of intermediate 5-amino-6-methoxybenzo[d] [1,3]oxathiol-2-one 4 with benzenesulfonyl chlorides. Characterization of the products was achieved by nuclear magnetic resonance (NMR) and electrospray ionization mass spectra (ESI-MS) techniques. Biological assay results have shown that most of compounds inhibited the main toxic activities of the venom of the two snake species. Compound 5b (N-(6-methoxy-2-oxobenzo[d] [1,3]oxathiol-5-yl)-4-nitrobenzenesulfonamide) was the most efficient in inhibiting hemolysis of B. jararaca, and coagulation and proteolysis induced by both venoms. For in vivo activities, all compounds inhibited the edema, from 35 to 72%, and most of them exhibited antihemorrhagic and antilethality activities. Thus, the results pointed to the biological potential of these compounds, being promising molecules to treat envenomation by these snakes as well as to aid the current antivenom serum therapy.


Introduction
Snake venoms are complex mixtures of proteins with or without enzymatic activity, including metalloproteinases, serine proteinases, phospholipases A 2 , and L-amino acid oxidase. According to World Health Organization (WHO), snakebite envenoming represents a public health problem worldwide and is considered a neglected disease. 1,2 In Brazil, the venomous pit vipers Bothrops are responsible for the majority of snakebites (90%), and B. jararaca and B. jararacussu are amongst the highest venomous species. B. jararaca is found in southern effects into victims, as early anaphylactic reactions and fever. 5,6 Thus, seeking for strategies to block toxic effects of snake venom is a considerable challenge, and natural products have been investigated, elsewhere. 7 On the other hand, molecules derived from organic synthesis have not been widely investigated. In this context, heterocycles play an essential role in drug design, since they comprise a class of substances of great synthetic interest due to their occurrence in natural products and pharmacologically active compounds. [8][9][10][11][12] In particular, 1,3-benzoxathiol-2one and its derivatives have been found to possess diverse biological activities, such as antibacterial, antifungal, antioxidant, anticancer, anti-inflammatory and carbonic anhydrase and monoamine oxidase (MAO) enzymes inhibitors. [13][14][15][16][17][18] To the best of our knowledge, the antivenom effect of such class of molecules has not been studied. On the other hand, sulfonamide derivatives have already been reported for their ability to neutralize the hemolytic activity of the venom of Lachesis muta, 19 as well as to inhibit some in vitro and in vivo toxic effects of the venoms of B. jararaca or L. muta. 20 Besides, drugs derived from sulfa have been utilized in some pharmaceutical applications due to their antibacterial, antiviral, antimalarial, antifungal, anticancer, antidepressant and other properties. 21,22 In continuation of our efforts on the synthesis of new biologically active 1,3-benzoxathiol-2-one-based compounds, we herein report the synthesis of new 1,3-benzoxathiol-2-one sulfonamides and evaluation of their ability to inhibit some toxic in vitro and in vivo activities of B. jararaca and B. jararacussu venoms.

Chemistry
Reactions were routinely monitored by thin-layer chromatography (TLC) on silica-gel precoated F 254 Merck plates (Darmstadt, Germany) visualized under UV light (254-366 nm). Melting points were determined on a Fisatom 430 apparatus (São Paulo, Brazil) and are uncorrected. Catalytic hydrogenation reaction was performed on a Paar 4540 reactor (Moline, USA). Infrared (IR) spectra were recorded on a PerkinElmer 1420 spectrometer (Waltham, USA) using KBr pellets and frequencies are expressed in cm -1 . Electrospray ionization mass spectra (ESI-MS) in the negative ion mode were recorded on a Waters ZQ-4000 single quadrupole mass spectrometer (Milford, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity Plus 300 and 500 (Palo Alto, USA) spectrometers in dimethyl sulfoxide (DMSO-d 6 ), which was used as the deuterated solvent (Cambridge Isotope Laboratories Inc., Tewksburry, USA). Chemical shifts (d) are reported in parts per million (ppm) relative to tetramethylsilane (TMS), which was used as an internal standard. All reagents and solvents were used as obtained from commercial suppliers without further purification. In addition, pyridine, ethyl acetate, hydrochloric acid, diethyl ether and magnesium sulfate were purchased from Vetec Química Fina LTDA (Duque de Caxias, Brazil). Finally, 6-hydroxy-benzo[d] [1,3] oxathiol-2-one, 4-substituted benzenesulfonyl chlorides and 4-(dimethylamino)pyridine (DMAP) were obtained from Sigma-Aldrich (São Paulo, Brazil).
Procedures for preparing 1,3-benzoxathiol-2-one derivatives 2, 3 and 4 Protocols for the preparation, physical and spectroscopic data of compounds 2, 3 and 4 have already been reported in our previous papers. [13][14][15] General procedure for the synthesis of sulfonamides (5a-5h) In a round-bottom flask, pyridine (10 mL), 5-amino-6-methoxybenzo[d] [1,3]oxathiol-2-one (4) (1 mmol), the appropriate 4-substituted benzenesulfonyl chloride (1.5 mmol) and DMAP (1 mmol) were added. The system was heated at reflux for 8 h. Then, the solution was neutralized with concentrated HCl. The reaction mixture was extracted with ethyl acetate (4 × 20 mL), the combined organic layers were dried over MgSO 4 and evaporated under reduced pressure. The oil obtained was triturated with diethyl ether to afford a solid product. After completing the crystallization, the solid was filtered and was washed with hot water followed by diethyl ether. [1,3]

Venoms, animals, and reagents
Bothrops jararaca and B. jararacussu venoms were kindly supplied by Fundação Ezequiel Dias (FUNED), Belo Horizonte, Minas Gerais State, Brazil, and stored at -20 °C until use. The collection of snake venom was conducted under the authorization of the Brazilian System for Management Genetic of Heritage and Associated Traditional Knowledge (SisGen) (process number A39CD4E). Balb/c mice (18-20 g) were obtained from the Laboratory Animal Care of the Federal Fluminense University (UFF) and were housed under constant temperature (24 ± 1 °C) and light conditions. Experiments were approved by the UFF Institutional Committee for Ethics in Animal Experimentation (protocol number 508), that are in accordance with the guidelines of the Brazilian Committee for Animal Experimentation (COBEA). All solvents or reagents were of the best grade available.

Biological assays
Coagulant activity of venoms Different concentrations of B. jararaca or B. jararacussu venom (5-70 μg mL -1 ) were added to plasma and coagulation time was monitored using a digital Amelung coagulometer (model KC4A, Labcon, Germany). The amount of venom (μg mL -1 ) able to clot plasma around 60 s was called minimum coagulation dose (MCD), and such venom concentration (B. jararaca, 35 μg mL -1 or B. jararacussu, 50 μg mL -1 ) was incubated for 30 min at 25 °C with compounds, at 1:10 venom:compound ratio (m/m). After incubation, mixture was added to the medium reaction and coagulation was monitored, as described. Control experiments were performed by incubating compounds, dimethyl sulfoxide (DMSO) (0.8%, final concentration) or saline with plasma in the absence of venoms.

Proteolytic activity of venoms
Proteolytic activity of B. jararaca and B. jararacussu venom was determined 23 using azocasein as a substrate (0.2% m/v, in 20 mM Tris-HCl, 8 mM CaCl 2 , pH 8.8), with modifications. Different concentrations of each venom (2-40 μg mL -1 ) were incubated with azocasein, and the amount of each venom that achieved supramaximal activity was considered as 100% of proteolytic activity, and called effective concentration (EC). The effect of compounds was evaluated by incubating one EC of B. jararaca or B. jararacussu (10 μg mL -1 ) venom with compounds (100 μg mL -1 ) for 30 min at 25 °C. Then, proteolytic activity was determined, as described. Control experiments were performed by incubating compounds, DMSO (0.9%, final concentration) or saline, instead of venoms.

Hemolytic activity of venoms
Hemolytic activity of the venom of B. jararacussu was not performed, because of the low hemolytic activity of this venom. On the other hand, the degree of hemolysis caused by the venom of B. jararaca was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion, as substrate. 24 After performing a concentration-response curve (5-50 μg mL -1 ), the amount of venom (μg mL -1 ) able to induce 100% of hemolysis was called minimum indirect hemolytic concentration (MIHC). Then, one MIHC of B. jararaca (24 μg mL -1 ) venom was incubated with compounds (240 μg mL -1 ) or solvents (DMSO or saline) for 30 min at 25 °C, followed by the hemolytic test. Control experiments were performed by incubating venom with solvents in the absence of compounds or by adding solely compounds or solvents to reaction medium.

Hemorrhagic activity of venoms
Hemorrhagic lesions produced by B. jararaca and B. jararacussu venoms were quantified by the procedure described by Kondo et al., 25 with modifications. B. jararaca or B. jararacussu venom was injected intradermally (i.d.) into the abdominal skin of mice, and 2 h later, animals were euthanized by decapitation, abdominal skin removed, stretched, and inspected for visual changes in the internal aspect in order to localize hemorrhagic spots. One minimum hemorrhagic dose (MHD) was defined as the amount of venom (µg per mice) able to produce a hemorrhagic halo of 10 millimeters that was 12 µg per mice. The effect of compounds on venom-induced hemorrhage was investigated by incubating compounds with two MHD of B. jararaca or B. jararacussu venom (24 µg per mice) for 30 min at 25 °C. After incubation, mixture was injected i.d. into mice, and hemorrhagic activity was performed, as described. Negative controls were performed by injecting solely saline, DMSO or compounds, instead of venom. The volume of injection of samples was 100 μL.

Edematogenic activity of venoms
Edema-inducing activity of venom was determined in accordance to Yamakawa et al.,26 with modifications. Groups of five mice received a subcutaneous (s.c.) subplantar single injection of B. jararaca or B. jararacussu venom into the right paw; while the left one received a single injection of saline or DMSO. Then, 1 h after injection, the paws were amputated, weighed and edema was evaluated and expressed as the percentage of increase in the weight of the right foot paw compared to the left one. The effect of compounds was evaluated by incubating each venom (7 µg per mice) with compounds (20 µg per mice) for 30 min at 25 °C, and then, an aliquot of this mixture was injected s.c. into mice. Control experiments were performed by injecting compounds or solvents in the absence of venom. The volume of injection of samples was 50 μL.
Lethality activity of venoms B. jararaca or B. jararacussu venom (50 μg per mice) was incubated with solvents for 30 min at 25 °C, and an aliquot was injected intraperitoneally (i.p.) into mice, and deaths were observed and registered. Antilethality effect was performed by incubating each venom with compounds (150 μg per mice) at the same conditions. After incubation, mixture was injected i.p. into mice, and time of death was observed, and compared to the group that received injection of venoms alone. Negative control group received injection solely of compounds or solvents, instead of venom. After the end of injections, number of deaths of mice was observed over a period of 24 h. The volume of injection of samples was 100 μL.

Statistical analysis
Results are expressed as means ± standard deviation (SD). The statistical significance of differences amongst experimental groups was evaluated using the Student's t-test. p values < 0.05 were considered significant.
Sulfonylation reaction between the amine 4 and different 4-substituted benzenesulfonyl chlorides was attempted with other methodologies by varying solvent, base, catalyst and temperature, as shown in Table 1. The protocols presented in entries 1 to 9 were unsuccessful. It is noteworthy the comparison between entries 9 and 10, which corroborates with a proposal that, in this reaction, the DMAP plays a dual role as a base and nucleophilic catalyst, and therefore can activate both reactants in this reaction (Figure 1). 27 The structures of the newly synthesized compounds were characterized by spectral data ( 1 H NMR, 13 C NMR, IR and ESI-MS). 2D-NMR techniques (correlated spectroscopy (COSY), heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond correlation (HMBC)) helped us to assign the correct signals of the compounds. The results of the spectral analysis were in accordance with the proposed structures. As an example, the 1 H NMR spectrum of compound 5a exhibited a broad singlet at 9.82 ppm for N-H. Hydrogens H7 and H4 appeared as singlets at 7.64 and 7.16 ppm, respectively. Hydrogens H2'/H6' and H3'/H5' are shown as duplets at 7.57 ppm (J 8.0 Hz) and 7.32 ppm (J 8.0 Hz), respectively. Two singlets were identified at 3.46 and 2.35 ppm for O−CH 3 and CH 3 groups, respectively. The 13 C NMR spectrum exhibited the C=O signal at 169.7 ppm, OCH 3 at 56.1 ppm and the CH 3 at 20.9 ppm. IR spectrum of 5a showed the N−H and C=O stretching vibrations at 3258 and 1768 cm −1 , respectively. The bands at 1336 and 1161 cm −1 represent sulfur-oxygen stretching frequencies in sulfone group (SO 2 ).

Biological assays
The inhibitory ability of the synthesized compounds (5a-5h) against some activities of the venoms of B. jararaca and B. jararacussu was investigated. The tested activities, coagulant, proteolytic, hemolytic, Scheme 1. Synthetic route used for preparing 5a-5h. i: HNO 3 65%, CH 2 Cl 2 , r.t., 2 h; ii: CH 3 I, K 2 CO 3 , DMF, r.t., 24 h; iii: H 2 , 10% Pd/C, EtOH, 20 bar, 50 °C, 6-8 h; iv: p-ArSO 2 Cl, pyridine, DMAP (1 equiv.), reflux, 8 h.  hemorrhagic, edematogenic, and lethality are responsible for the most important symptoms after snakebites, and cause into victims many toxic effects, leading to morbidities, amputation or deaths. Thus, it is essential to evaluate such activities of venoms to develop a candidate molecule as antivenom. It is worth mentioning that a similar ratio between B. jararaca or B. jararacussu venom and the compounds was used for most of all the in vitro and in vitro assays, that it was of 1:10 venom:compound (m/m). This strategy was employed to compare better the efficacy of neutralization of all compounds in each assay in this article as well as previous results of our group. 19,20 Effect of compounds on in vitro activities of B. jararaca and B. jararacussu venoms

Anticoagulant activity
Snake venoms are composed of many enzymes, which are responsible for toxic effects observed in victims. The snake venom serine proteases (SVSPs) and snake venom metalloproteases (SVMPs) enzymes are the most important and majority group of enzymes. They act on a variety of tissues and systems. SVSPs alter blood coagulation system, and most of them have thrombin-like activity, leading to the formation of fibrin, and thus, promoting the formation of thrombus. On the other hand, SVSPs may induce hemorrhage of victims because of the high consumption of fibrinogen, impairing blood to clot. In contrast, the SVMPs induce hemorrhage and coagulation of blood. Snakebites caused by the species of Bothrops genus provoke tissue necrosis and intravascular thrombin formation, resulting in coagulopathies and hemorrhage. However, B. jararaca venom is more hemorrhagic than B. jararacussu venom, due to presence of the SVMPs jararhagin 28 and bothropasin, 29 previously isolated from B. jararaca venom. On the other hand, B. jararacussu venom is more myotoxic due to the action of two myotoxins with phospholipase A 2 (PLA 2 ) structure, bothropstoxin I (Bthtx-I, without catalytic activity) and bothropstoxin-II (Bthtx-II, with catalytic activity). 30,31 Thus, a molecule able to impair snake venom-induced coagulation, proteolysis or hemorrhage is relevant.

Antiproteolytic activity
B. jararaca and B. jararacussu venoms are rich sources of proteases, and each venom was able to hydrolyze the chromogenic substrate, azocasein. Then, 100 μg mL -1 of compounds (5a-5h) were incubated with each venom (10 μg mL -1 ), as described in Experimental section. As shown in Figure 3, compounds 5b (39%) and 5h (27%) were the most efficient to inhibit proteolytic activity of B. jararacussu and B. jararaca venom, respectively. On the other hand, the compounds 5d-5g did not inhibit proteolysis of venoms ( Figure 3). None of the compounds solely had proteolytic activity.

Antihemolytic effect
Hemolytic activity was not performed for the venom of B. jararacussu because it had low hemolytic activity. The concentration of B. jararaca venom (24 μg mL -1 ) that induced 100% of lysis of red blood cells was called minimum indirect hemolytic concentration (MIHC). This MIHC of venom (24 μg mL -1 ) was incubated with 240 μg mL -1 of compounds (5a-5h), and then hemolytic activity was performed, as described in Experimental section. As shown in Figure 4, the compounds 5b and 5h inhibited around 55%, while the compounds 5d and 5e inhibited approximately 20%. The compounds 5a, 5c, 5f and 5g did not inhibit hemolysis of B. jararaca venom. Hemolytic activity of snake venoms is due to the participation of another group of enzymes, phospholipase A 2 (PLA 2 ). Beyond hemolysis, PLA 2 enzymes inhibit platelet aggregation, induce edema or myotoxicity, and, thus, participate in the development of tissue necrosis. None of the compounds solely induced hemolysis (data not shown).
It is noteworthy that the most active compound in inhibiting hemolysis of B. jararaca, and coagulation and proteolysis induced by both venoms, (N-(6-methoxy-2-oxobenzo[d] [1,3]oxathiol-5-yl)-4-nitrobenzenesulfonamide) (5b), bears the nitro group in its structure. The nitro group has a strong electron-withdrawing ability that creates localized or regional electron deficient sites within molecules, allowing interactions with biological nucleophiles present in living systems, such as proteins, amino acids, nucleic acids, and enzymes. The interaction may occur by a nucleophilic addition or displacement, by electron transfer involving oxidation and reduction, or simply by molecular complexation. 32,33 This fact may suggest the importance of the nitro group for the biological activity. Among the halogenated compounds, the most active to prevent B. jararaca venom-induced hemolytic activity was the 4-iodo-N-(6-methoxy-2-oxobenzo[d] [1,3]oxathiol-5-yl)benzenesulfonamide (5h), which has an iodine atom on the benzene ring. Interestingly, a decrease in the inhibitory effect was observed with an increasing of halogen atom electronegativity, in the order, compound 5h (iodine), 5d (bromine), 5e (chlorine), 5c (fluorine). It is known that aromatic rings, such as benzene and its derivatives, act as ligands (Lewis base) in a variety of complexes, 34 and the greater the electron density in the ring, greater the reactivity towards the metal cation, such as Ca 2+ , an ion that stimulate the activity of some PLA 2 enzymes. It was observed that compound 5c did not inhibit hemolysis of B. jararaca venom. The presence of a fluorine atom, the most electronegative among halogens, may have contributed to the lack of activity. On the other hand, the iodine atom, the less electronegative, may have enabled a higher interaction between benzene ring and the Ca 2+ , through a cation-π interaction, 35 which corroborates with an inhibition around 55% for the compound 5h. Injection intradermically (i.d.) of B. jararaca or B. jararacussu (24 μg per mice) venom produced a hemorrhage halo of around 20 mm that was considered as 100% of hemorrhagic activity. This dose of venom, which represents 2 MHD, was incubated for 30 min at 25 ºC with the compounds (240 μg per mice). After incubation, the mixture was injected i.d. into mice, and hemorrhage was analyzed. As seen in Figure 5, all the compounds inhibited the hemorrhagic activity of venoms, but with different inhibitory percentages (from 6 to 62%). Thus, the  compounds interfered with SVMPs, maybe by chelating Zn 2+ , as they depend on this metal to display hemorrhagic activity. The compound 5f inhibited more efficiently B. jararaca venom-induced hemorrhage, probably due to its ability to bind to the main toxic enzymes of such venom, jararhagin 28 or bothropasin. 29 The compound 5a did not inhibit B. jararacussu-induced hemorrhage. Thus, one may speculate the inability of the compound 5a to bind to divalent cations (as Zn 2+ or Ca 2+ ), leading to an inefficacy to inhibit hemorrhagic ( Figure 5) or hemolytic (Figure 4) activity of B. jararaca or B. jararacassu venom. None of the compounds or solvents induced hemorrhage.
B. jararaca (black columns) or B. jararacussu (white columns) venom (24 μg per mice) was mixed with 240 μg per mice of compounds (5a-5h) for 30 min at 25 °C. Then, mixture was injected i.d. into mice, and hemorrhagic activity was analyzed, and expressed as inhibition of hemorrhage.

Antiedematogenic activity
Injection subcutaneously (s.c.) of B. jararaca or B. jararacussu venom (7 µg per mice) into the paw of mice produced an increase of 40% on their paw volume, and such increase was considered as 100% of edematogenic activity. After, venom (7 µg per mice) was incubated with the 20 μg per mice of compounds (5a-5h) for 30 min at 25 °C. Then, mixture was injected s.c. into the paw of mice, and edema was analyzed. All compounds inhibited edema of B. jararaca and B. jararacussu venoms, from 35 to 72% ( Figure 6). PLA 2 enzymes of venoms may contribute to edematogenic effect of venoms, as well. The PLA 2 enzymes need Ca 2+ to display such activity, and the negatively charged structure of some compounds might bind to the active site of PLA 2 enzymes and inhibit hemolytic activity or any other related toxic activity related to such enzymes, as edema. Neither the compounds nor solvents (saline or DMSO) induced edema.
B. jararaca or B. jararacussu venom (7 μg per mice) was mixed with 20 μg per mouse of compounds (5a-5h) for 30 min at 25 ºC. Then, mixture was injected s.c. into mice and edematogenic activity was evaluated, as described, and inhibition of edema was determined.

Antilethality activity
A single intraperitoneal (i.p.) injection of 13 μg per mice of B. jararaca or B. jararacussu venom incubated with saline or DMSO killed mice approximately in 65 min. As seen in Table 2, 120 μg per mice of the compounds (5a-5h) protected mice of the lethality of B. jararacussu venom. On the other hand, the compounds 5a, 5f and 5g failed to protect mice of death caused by B. jararaca venom. Nevertheless, the other compounds did impair the death of mice induced by such venom. Injection of solely compounds did not cause death of mice, even at higher concentrations (up to 200 μg per mice). The maximal time of observation of the survival of mice was 300 min.
The compounds 5a-5h (120 μg per mice) were incubated for 30 min at 25 °C with B. jararaca or B. jararacussu venom (13 μg per mice). Then, the mixture was injected i.p. into mice, and survival time was monitored. Control experiments were performed by incubating B. jararaca or B. jararacussu venom with saline or DMSO. The total time of observation of survival time of mice was 300 min.

Conclusions
In summary, a series of eight novel 1,3-benzoxathiol-2one sulfonamide derivatives was successfully synthesized and characterized by IR, 1 H and 13 C NMR, and ESI-MS  analysis. These compounds were able to inhibit some of the main toxic activities of the venom of B. jararaca or B. jararacussu, but with different potencies. Thus, being promising molecules to treat envenomation by these snakes as well as to aid the current antivenom serum therapy. However, it is quite difficult to postulate a mechanism of action of compounds based on their chemical structure, because snake venoms are a complex mixture of toxic proteins acting through unknown mechanisms of action.

Supplementary Information
Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.