Efficient Catalytic Oxidation of 3-Arylthio- and 3-Cyclohexylthio-lapachone Derivatives to New Sulfonyl Derivatives and Evaluation of Their Antibacterial Activities

New sulfonyl-lapachones were efficiently obtained through the catalytic oxidation of arylthio- and cyclohexylthio-lapachone derivatives with hydrogen peroxide in the presence of a Mn(III) porphyrin complex. The antibacterial activities of the non-oxidized and oxidized lapachone derivatives against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus were evaluated after their incorporation into polyvinylpyrrolidone (PVP) micelles. The obtained results show that the PVP-formulations of the lapachones 4b–g and of the sulfonyl-lapachones 7e and 7g reduced the growth of S. aureus.

Since derivatives containing sulfur groups are interesting compounds due to their significant properties as intermediates in many biological processes [28,29], our group recently reported a straightforward and efficient one-step reaction to prepare new nor-β-lapachone derivatives (4) tethered with thio-substituents at position 3 of the furan ring ( Figure 1) [30]. It is known that sulfones are widely used as solvents, polymers, and biopharmaceutical agents. Indeed, several drug molecules containing sulfone groups are used for the treatment of leprosy, dermatitis herpetiformis, and tuberculosis. Moreover, sulfones have also demonstrated various biological activities, mainly as anti-inflammatory, antimicrobial, anticancer, anti-HIV, antimalarial, and anti-inflammatory [31] properties. In particular, sulfones conjugated to naphthoquinones have several biological activities reported in the literature. Lee and coworkers [32] reported a new series of naphthoquinone derivatives in which compound 5, (Figure 2), proved to be a most potent inhibitor against HepG2 cell lines (IC50 of 0.44 µM). Considering that the oxidation of organosulfur compounds can provide new derivatives with potential biological activities, and following our previous studies on oxidative transformations, we decided to evaluate the possibility of using a metalloporphyrin as a catalyst and hydrogen peroxide as oxidant in the oxidation process of 3-arylthio-nor-β-lapachone derivatives 4a-f. This aryl-substituted lapachone series was chosen keeping in mind the biological properties played by the aryl-substituted lapachones 2 and 3. However, in this work a cycloalkyl derivative, the cyclohexylthio-nor-β-lapachone 4g, was also considered to see if the alkyl-type substituent would give any significative difference in the biological assessment.

Synthesis of Sulfonyl-nor-β-lapachone Derivatives 7a-g
The synthetic strategy to obtain the new nor-β-lapachone derivatives 7a-g involved the experimental work summarized in Scheme 1. It is known that sulfones are widely used as solvents, polymers, and biopharmaceutical agents. Indeed, several drug molecules containing sulfone groups are used for the treatment of leprosy, dermatitis herpetiformis, and tuberculosis. Moreover, sulfones have also demonstrated various biological activities, mainly as anti-inflammatory, antimicrobial, anticancer, anti-HIV, antimalarial, and anti-inflammatory [31] properties. In particular, sulfones conjugated to naphthoquinones have several biological activities reported in the literature. Lee and coworkers [32] reported a new series of naphthoquinone derivatives in which compound 5, (Figure 2), proved to be a most potent inhibitor against HepG2 cell lines (IC 50 of 0.44 µM). It is known that sulfones are widely used as solvents, polymers, and biopharmaceutical agents. Indeed, several drug molecules containing sulfone groups are used for the treatment of leprosy, dermatitis herpetiformis, and tuberculosis. Moreover, sulfones have also demonstrated various biological activities, mainly as anti-inflammatory, antimicrobial, anticancer, anti-HIV, antimalarial, and anti-inflammatory [31] properties. In particular, sulfones conjugated to naphthoquinones have several biological activities reported in the literature. Lee and coworkers [32] reported a new series of naphthoquinone derivatives in which compound 5, (Figure 2), proved to be a most potent inhibitor against HepG2 cell lines (IC50 of 0.44 µM). Considering that the oxidation of organosulfur compounds can provide new derivatives with potential biological activities, and following our previous studies on oxidative transformations, we decided to evaluate the possibility of using a metalloporphyrin as a catalyst and hydrogen peroxide as oxidant in the oxidation process of 3-arylthio-nor-β-lapachone derivatives 4a-f. This aryl-substituted lapachone series was chosen keeping in mind the biological properties played by the aryl-substituted lapachones 2 and 3. However, in this work a cycloalkyl derivative, the cyclohexylthio-nor-β-lapachone 4g, was also considered to see if the alkyl-type substituent would give any significative difference in the biological assessment.

Synthesis of Sulfonyl-nor-β-lapachone Derivatives 7a-g
The synthetic strategy to obtain the new nor-β-lapachone derivatives 7a-g involved the experimental work summarized in Scheme 1. Considering that the oxidation of organosulfur compounds can provide new derivatives with potential biological activities, and following our previous studies on oxidative transformations, we decided to evaluate the possibility of using a metalloporphyrin as a catalyst and hydrogen peroxide as oxidant in the oxidation process of 3-arylthio-nor-β-lapachone derivatives 4a-f. This aryl-substituted lapachone series was chosen keeping in mind the biological properties played by the aryl-substituted lapachones 2 and 3. However, in this work a cycloalkyl derivative, the cyclohexylthio-nor-β-lapachone 4g, was also considered to see if the alkyl-type substituent would give any significative difference in the biological assessment.

Synthesis of Sulfonyl-nor-β-lapachone Derivatives 7a-g
The synthetic strategy to obtain the new nor-β-lapachone derivatives 7a-g involved the experimental work summarized in Scheme 1.

Scheme 1.
Synthetic access to naphthoquinones 4a-g and the oxidation conditions used for their conversion to 7a-g.
The starting nor-β-lapachone-3-thio-derivatives 4a-f were obtained according to data in the literature [30] and the synthesis of 4g is described here. The steps involved the reaction of nor-lapachol (6) with bromine, followed by the in situ quenching of the cyclic cationic o-quinone methide intermediate with the adequate thiol derivatives.
The oxidation of derivatives 4a-g was performed by using the manganese (III) complex of 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin, Mn(TDCPP)Cl (8) as a catalyst and hydrogen peroxide as the oxidant (Scheme 1). All reactions were performed in acetonitrile at ambient temperature and the oxidant was added every 15 min to the reaction mixture in aliquots of 0.15 mmol. After 1 h of reaction, thin-layer chromatography (TLC) control showed the total or almost total consumption of each starting arylthio/cyclohexylthio-lapachones 4a-g and the formation of a more polar product. After the workup and purification of the reaction mixture by preparative TLC using CH2Cl2 as eluent, followed by a detailed spectroscopic analysis of the major compound in each case, it was possible to identify the nor-β-lapachone sulfonyl-products 7a-g, which were isolated in yields ranging from 78% to 86% (Table 1). The structures of all new derivatives were confirmed by 1 H-and 13 C-NMR, IR spectroscopy and high-resolution mass spectra (HRMS-ESI) (see Experimental Section and Supplementary Materials). In particular, the mass spectra of derivatives 7 show molecular ions containing 32 mass units higher than the corresponding molecular ions of their precursors 4. Additionally, in the 13 C-NMR spectrum the distinctive signal of C3 of each sulfone occurs at a higher chemical shift than the corresponding C3 of its precursor 4, this being in agreement with the deprotection present in the product sulfone ( Figure 3 shows the cases of 4e and 7e). None of the other signals were strongly affected. Scheme 1. Synthetic access to naphthoquinones 4a-g and the oxidation conditions used for their conversion to 7a-g.
The starting nor-β-lapachone-3-thio-derivatives 4a-f were obtained according to data in the literature [30] and the synthesis of 4g is described here. The steps involved the reaction of nor-lapachol (6) with bromine, followed by the in situ quenching of the cyclic cationic o-quinone methide intermediate with the adequate thiol derivatives.
The oxidation of derivatives 4a-g was performed by using the manganese (III) complex of 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin, Mn(TDCPP)Cl (8) as a catalyst and hydrogen peroxide as the oxidant (Scheme 1). All reactions were performed in acetonitrile at ambient temperature and the oxidant was added every 15 min to the reaction mixture in aliquots of 0.15 mmol. After 1 h of reaction, thin-layer chromatography (TLC) control showed the total or almost total consumption of each starting arylthio/cyclohexylthio-lapachones 4a-g and the formation of a more polar product. After the workup and purification of the reaction mixture by preparative TLC using CH 2 Cl 2 as eluent, followed by a detailed spectroscopic analysis of the major compound in each case, it was possible to identify the nor-β-lapachone sulfonyl-products 7a-g, which were isolated in yields ranging from 78% to 86% (Table 1). The structures of all new derivatives were confirmed by 1 H-and 13 C-NMR, IR spectroscopy and high-resolution mass spectra (HRMS-ESI) (see Experimental Section and Supplementary Materials). In particular, the mass spectra of derivatives 7 show molecular ions containing 32 mass units higher than the corresponding molecular ions of their precursors 4. Additionally, in the 13 C-NMR spectrum the distinctive signal of C3 of each sulfone occurs at a higher chemical shift than the corresponding C3 of its precursor 4, this being in agreement with the deprotection present in the product sulfone ( Figure 3 shows the cases of 4e and 7e). None of the other signals were strongly affected.

Incorporation of 3-Arylthio/Cyclohexylthio-nor-β-lapachone Derivatives 4a-g and Nor-β-lapachone Derivatives 7a-g into Polyvinylpyrrolidone Micelles
PVP is a water-soluble and non-toxic polymer, widely used to modify the water solubility of numerous biologically active compounds and also their pharmacokinetic and pharmacological activities [44]. In fact, PVP has been successfully applied in drug delivery of several antibiotics and antifungal agents, demonstrating high efficiency in the delivery of these drugs [45][46][47].
The micelles of PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g were prepared by mixing chloroform solutions of PVP and lapachones (10:1 w/w). The resulting solutions were stirred for 2 h at room temperature and then the chloroform was evaporated under a nitrogen atmosphere. After this procedure, all residues were dissolved in 2 mL of water giving rise to the micelle solutions of PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g.

Incorporation of 3-Arylthio/Cyclohexylthio-nor-β-lapachone Derivatives 4a-g and Nor-β-lapachone Derivatives 7a-g into Polyvinylpyrrolidone Micelles
PVP is a water-soluble and non-toxic polymer, widely used to modify the water solubility of numerous biologically active compounds and also their pharmacokinetic and pharmacological activities [44]. In fact, PVP has been successfully applied in drug delivery of several antibiotics and antifungal agents, demonstrating high efficiency in the delivery of these drugs [45][46][47].
The micelles of PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g were prepared by mixing chloroform solutions of PVP and lapachones (10:1 w/w). The resulting solutions were stirred for 2 h at room temperature and then the chloroform was evaporated under a nitrogen atmosphere. After this procedure, all residues were dissolved in 2 mL of water giving rise to the micelle solutions of PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g.
A standard concentration of both bacteria was plated on Mueller-Hinton agar (Liofilchem, Italy). The 6-mm discs (Liofilchem, Italy) were immersed in the solutions of the formulations PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g at concentrations of 1 mM (concentration of each lapachone derivative) and placed on the plates that were incubated overnight at 37 • C. After incubation, the diameters of the inhibition halos were measured. In order to control the toxicity of PVP, a control experiment with a solution of PVP (10 mg/mL) was also carried out for each bacteria strain.
The results of the preliminary antimicrobial activity tests show that E. coli growth is not inhibited by any one of the lapachone derivatives 4a-g and 7a-g, since no inhibitory halos were formed. However, that was not the case with S. aureus; the inhibition halos' features obtained are summarized in Table 2 and shown in Figure 4.
A standard concentration of both bacteria was plated on Mueller-Hinton agar (Liofilchem, Italy). The 6-mm discs (Liofilchem, Italy) were immersed in the solutions of the formulations PVP-arylthio/ cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g at concentrations of 1 mM (concentration of each lapachone derivative) and placed on the plates that were incubated overnight at 37 °C. After incubation, the diameters of the inhibition halos were measured. In order to control the toxicity of PVP, a control experiment with a solution of PVP (10 mg/mL) was also carried out for each bacteria strain.
The results of the preliminary antimicrobial activity tests show that E. coli growth is not inhibited by any one of the lapachone derivatives 4a-g and 7a-g, since no inhibitory halos were formed. However, that was not the case with S. aureus; the inhibition halos' features obtained are summarized in Table 2 and shown in Figure 4.  These results show that S. aureus presents sensitivity to arylthio/cyclohexylthio-lapachones 4b-g and sulfonyl-lapachones' 7e and 7g PVP formulations ( Figure 4 and Table 2). It is important to emphasize that, with the exception of 4a, all the other arylthio/cyclohexylthio-lapachones formulations were able to reduce the growth of S. aureus, with the larger inhibition halos obtained with 4d and 4f. However, with the sulfone derivatives, only compounds 7e and 7g have been demonstrated to maintain the anti-bacterial activity of S. aureus. These results show that S. aureus presents sensitivity to arylthio/cyclohexylthio-lapachones 4b-g and sulfonyl-lapachones' 7e and 7g PVP formulations ( Figure 4 and Table 2). It is important to emphasize that, with the exception of 4a, all the other arylthio/cyclohexylthio-lapachones formulations were able to reduce the growth of S. aureus, with the larger inhibition halos obtained with 4d and 4f. However, with the sulfone derivatives, only compounds 7e and 7g have been demonstrated to maintain the anti-bacterial activity of S. aureus.
Melting points were obtained on a Fischer Jones apparatus and are uncorrected. Analytical grade solvents were used. Reagents were purchased from Aldrich. Column chromatography was performed on silica gel 60 (Merck 230-400 mesh, Rio de Janeiro, Brazil). Yields refer to purified compounds obtained by chromatographic techniques and confirmed by spectroscopic data. Reactions were monitored by thin-layer chromatography (TLC) performed on 0.25-mm E. Merck silica gel plates (60F-254) using UV light as the visualizing agent. Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrum One spectrophotometer (Jardim das Laranjeiras, Brazil), calibrated relatively to the 1601.8 cm −1 absorbance of polystyrene. NMR spectra were recorded on a Varian Unity Plus VXR (500 MHz) instrument in DMSO-d 6 or CDCl 3 solutions. The chemical shift data are reported in units of δ (ppm) downfield from tetramethylsilane or the solvent, either of which were used as the internal standard.
Coupling constants (J) are reported in Hertz and refer to apparent peak multiplicities. High-resolution mass spectra (HRMS) were recorded on a mass spectrometer, MICROMASS Q-TOF (Waters, Barueri, Brazil).

Synthesis of 3-Cyclohexylthio-nor-β-lapachone (4g)
A round-bottom flask containing a solution of nor-lapachol (500 mg, 2.2 mmol) in 40 mL of dry chloroform was externally cooled with ice and under an inert atmosphere, 4.4 mL (13.2 g, 8.36 mol) of bromine was added. Immediately a red solid precipitate of the cationic ortho-quinone methide derivative was formed; the reaction mixture was left stirring for additional 10 min. The bromine excess was removed under reduced pressure and immediately chloroform (40 mL) was added; the mixture was kept externally cool in an ice bath. To that mixture, chloroform solution with cyclohexanethiol (511 mg, 4.4 mmol in 25 mL) was slowly added. The reaction mixture was stirred for another 3 h and then poured into distilled water (50 mL). The organic phase was collected and successively washed with aqueous sodium bicarbonate solution (3 × 50 mL), and distilled water (3 × 50 mL), dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The resulting red solid was taken in chloroform and was purified by silica gel flash column chromatography, eluting with gradient mixtures of hexane and ethyl acetate.

General Procedure for the Synthesis of 3-Aryl/Cyclohexyl-sulfonyl-nor-β-lapachone Derivatives (7a-g)
For the catalytic studies, each substrate of 4a-g (0.3 mmol), the catalyst (2.0 × 10 −3 mmol, where the sub/cat molar ratio used was 150) and the co-catalyst (ammonium acetate, 0.2 mmol) were dissolved in CH 3 CN (2.0 mL). The reaction mixtures were kept under magnetic stirring and in the absence of light at 22-25 • C. The oxidant, 30% H 2 O 2 (w/w, aqueous solution), was diluted with CH 3 CN (1:10) and this was followed by the addition of 0.15 mmol of the oxidant taking place at every 15 min. The reactions were followed by TLC. For all the substrates, the resulting sulfones were isolated by preparative TLC using CH 2 Cl 2 as eluent.    3.4. General Procedure for the Incorporation of 3-Arylthio/cyclohexylthio-nor-β-lapachone Derivatives 4a-g and 3-Aryl/Cyclohexyl-sulfonyl-nor-β-lapachone 7a-g Derivatives into PVP Micelles To chloroform solutions of PVP (20 mg in 2 mL), a chloroform solution of ca. 2 mg of each 3-arylthio/cyclohexylthio-nor-β-lapachones 4a-g or 3-aryl/cyclohexyl-sulfonyl-nor-β-lapachone 7a-g (in 2 mL of chloroform) was added. The resulting solutions were stirred for 2 h at room temperature and then the chloroform was evaporated under nitrogen atmosphere. In order to remove all the organic solvents, the residues were kept in an oven at 45 • C for 24 h. After this procedure, all residues were dissolved in 2 mL of water, leading to the aqueous solution of PVP-arylthio/cyclohexylthio-lapachones 4a-g and PVP-sulfonyl-lapachones 7a-g micelles.
The antibacterial evaluation of PVP formulations of 3-arylthio/cyclohexylthio-nor-β-lapachone derivatives 4a-g and 3-sulphonyl-nor-β-lapachones 7a-g was done according to the European Committee on Antimicrobial Susceptibility Testing standards (EUCAST 2015). The bacterial cultures of S. aureus and E. coli cultivated in TSB were diluted 1:100 in 0.85% saline solution to obtain a density of 0.5 MacFarland. After that, for each bacterium a sterile cotton swab was dipped into the suspension and the inoculum was spread over the entire surface of a Muller-Hinton plate by swabbing in three directions. Then, sterilized disks were immersed into the compounds with a concentration of 1 mM and placed at the plate and incubated inverted at 37 • C for 16-20 h. The diameters of inhibition zones were measured.

Conclusions
Herein we present a new, efficient and environmentally friendly methodology, involving the use of a manganese (III) porphyrin complex as a catalyst and aqueous hydrogen peroxide as an oxidant in order to promote the oxidation of several organosulfur derivatives of nor-β-lapachone. This methodology allowed the preparation of the new sulfonyl-lapachones 7a-g in excellent yields through the oxidation of arylthio/cyclohexylthio-lapachones 4a-g.
For the antibacterial activity studies, the lapachone derivatives were successfully incorporated in polyvinylpyrrolidone (PVP) micelles. The PVP-arylthio/cyclohexylthio-lapachones and PVP-sulfonyllapachones' micelles were tested against a Gram-positive (S. aureus) and a Gram-negative (E. coli) bacteria. The preliminary results showed that such formulations are not active against E. coli. However, the PVP formulations with arylthio/cyclohexylthio-lapachones 4b-g and with sulfonyl-lapachones 7e and 7g reduced the growth of S. aureus. These compounds can be considered as prototypes for future antibacterial agents.