A New Kind of Quinonic-Antibiotic Useful Against Multidrug-Resistant S. aureus and E. faecium Infections

A rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics and reducing the therapeutic arsenal available for treatment of infectious diseases. In the present study, we developed a new class of compounds with antibacterial activity obtained by a simple, two step synthesis and screened the products for in vitro antibacterial activity against ATCC® strains using the broth microdilution method. The compounds exhibited minimum inhibitory concentrations (MIC) of 1–32 μg/mL against Gram-positive ATCC® strains. The structure–activity relationship indicated that the thiophenol ring is essential for antibacterial activity and the substituents on the thiophenol ring module, for antibacterial activity. The most promising compounds detected by screening were tested against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) clinical isolates. We found remarkable activity against VREF for compounds 7 and 16, were the MIC50/90 were 2/4 µg/mL and 4/4 µg/mL, respectively, while for vancomycin the MIC50/90 was 256/512 µg/mL. Neither compound affected cell viability in any of the mammalian cell lines at any of the concentrations tested. These in vitro data show that compounds 7 and 16 have an interesting potential to be developed as new antibacterial drugs against infections caused by VREF.


Introduction
Infectious diseases are a leading cause of death worldwide, and the increasing emergence of antibacterial resistance has contributed to rising rates of potentially fatal infections. It is estimated that by 2050, diseases caused by antibiotic-resistant microorganisms will be responsible for 10 million deaths per year [1]. The bacteria implicated in these infections include the so-called ESKAPE pathogens, such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and various Enterobacter sp. [2,3]. These microorganisms have become a global public health problem due to their ability to adapt to antibacterial agents. S. aureus-related skin infections, wound infections, and sepsis cases are the leading causes of healthcare-associated infections [4], and about 90% of S. aureus isolates were found to be methicillin-resistant in a study using data from 21 of the 35 countries in the Americas [4]. In the United States (US), these resistant strains are responsible for over 11,000 deaths per year [5]. In addition, Enterococcus species cause significant numbers of urinary tract, surgical site, and blood infections [4]. In the United Kingdom [6] and the US [7], up to 25% and 60% of E. faecium strains are resistant to vancomycin (VAN), respectively.
Traditionally, development of new antibacterial molecules has been based mainly on two strategies: modifying or adding a small chemical group to an antibiotic already in clinical use, to improve some aspect of its pharmacodynamic and/or pharmacokinetic profile; or seeking new molecules from natural products such as plants, bacteria, or fungi that have demonstrated activity against resistant bacteria. Both strategies involve structural modifications or additions that preserve the pharmacophore and therefore maintain both the mechanism and site of action. Optimizing these compounds may be initially effective, however, due to the structural similarity between novel and existing molecules, bacteria rapidly adapt their resistance mechanisms to thwart new antibiotics [8]. The traditional chemical approach is effective for identifying and optimizing compounds to treat pathologies such as hypertension, diabetes, dyslipidemia, inflammation, and allergies, in which the pharmacological targets do not adapt or generate resistance mechanisms. Infections, however, pose a different challenge, as bacteria are free-living organisms that seek to survive in the presence a harmful agent.
If we continue to rely exclusively on these traditional strategies, it is only a matter of time before our entire investment in the generation of antibiotics is overwhelmed by antibacterial resistance. The way forward should focus on rational, design-oriented development of new synthetic molecules capable of reducing the probability that exposed bacteria will generate a resistance phenotype. Antibacterial agents that have novel chemical structures and that act on unexplored bacterial targets are less likely to be subject to existing compound-or target-based resistance mechanisms. Of course, even new classes of antibiotics may be subject to general mechanisms of resistance, such as increased efflux, reduced influx, or target-site resistance mutations [8].
New approaches should consider a target that is different from existing targets, essential for microbial cell survival, highly conserved in clinically relevant species, absent or radically different in human cells, and easy to assay and approach biochemically [9,10]. However, structural modifications based on the traditional medicinal chemistry approach will also be needed to optimize effectiveness, and rational design will require synthesis of multiple compounds in order to determine the relationship between structure and activity. Therefore, it is critical to use a simple, versatile, and low-cost process to synthesize these molecules.
In this regard, various activities have been attributed to quinonic compounds. These compounds present two important characteristics for drug design. First is the versatility of synthetic processes, which allows active compounds to be obtained in a few stages; the second is the broad spectrum of biological activities described, which shows that the choice of substituents is critical to guiding the objective of biological activity, as shown by the work of Gordaliza et al. [11]. Quinonic compounds exert interesting antibacterial effects [12][13][14] and have already been integrated into antibacterial compounds, such as alkannin [15] and renierone [16]. These quinone-based antibiotics have been found to have activity against S. aureus, E. faecium, and Bacillus subtilis [17,18]. Promisingly, Tandon et al. have shown that thioaryl substitution in naphtoquinone results in good antibacterial activity [19,20]. However, it has not been further studied how the substituents of quinone compounds are related to their antibacterial activity. This information can be used to guide the rational design of new antibacterial quinone compounds.
In this study, our group develop a new kind of antibacterial agent based structurally on the quinonic core. We synthesized and assessed a set of 17 compounds against American Type Culture Collection (ATCC ® ) bacterial strains and addressed the study of the structure-activity relationship based on lipophilicity (logP), half wave potential (E 1/2 ), and volume (MR) parameters. We also analyzed the crystallographic structure of the two compounds with the best antibacterial performance and tested their activity against multidrug-resistant clinical isolates, to calculate minimum inhibitory concentration (MIC) 50/90 and minimum bactericidal concentration (MBC) 50/90 values against S. aureus and E. faecium. Finally, the toxicity of these two compounds against the HeLa, HTC-116, SHSY-5Y, and Vero cell lines was evaluated to assess their safety and suitability for use as a treatment.

Synthesis and Determination of Physicochemical Parameters
The final compounds were formed in two steps. First, we used activated benzoquinone, generated in situ from the corresponding hydroquinone with silver (I) oxide, reacting with enaminone through an ionic [3+3] process to produce the corresponding core 1 with a yield of 84% by a further in situ oxidation. During the second step, compounds 2-17 were obtained by nucleophilic addition of the corresponding thiophenol derivatives to core 1 with subsequent aerobic oxidation of the hydroquinone formed by Michael addition (Figure 1). Yields ranged from 47% to 88% for the 2 -, 3 -, and 4 -series. The lipophilic (LogP), half-wave potential (E 1/2 ) and molar refractivity (MR) parameters (Table 1) are discussed in Section 2.2. It should be noted that formation of the final products was highly favorable, with reactions produced under mild reaction conditions at room temperature. The total production time for each compound was 48 h. In addition, each product had a characteristic color that was distinct from that of the starting agent (yellow), facilitating purification by enabling rapid visual detection during the column chromatographic separation.

Antibacterial Screening
The antibacterial profile shows that all compounds except 5 and 6 demonstrated activity against Gram-positive bacteria (methicillin-resistant S. aureus [MRSA] ATCC ® 43300, methicillin-susceptible S. aureus [MSSA] ATCC ® 29213, and E. faecalis ATCC ® 29212). No activity was observed against Gram-negative bacteria (Escherichia coli ATCC ® 25922 and P. aeruginosa ATCC ® 27853), even at the highest concentrations tested (32 µg/mL, maximum solubility for the compounds). The antibacterial screening results are presented in Table 1. However, preliminary tests showed that adding EDTA to compounds [21] may induce activity against Gram-negative bacteria (unpublished results), through two possible mechanisms: fluidizing the lipid membrane [21] to facilitate passage of the derivatives through the membrane; or enabling the compound to act as a siderophore, in which the compound takes advantage of the high affinity of the iron transport system to facilitate entry into the bacterium [22]. This result suggests that it is possible to achieve activity against Gram-negative bacteria using a strategy that enables penetration of the bacterial membrane.

Antibacterial Screening
The antibacterial profile shows that all compounds except 5 and 6 demonstrated activity against Gram-positive bacteria (methicillin-resistant S. aureus [MRSA] ATCC ® 43300, methicillin-susceptible S. aureus [MSSA] ATCC ® 29213, and E. faecalis ATCC ® 29212). No activity was observed against Gramnegative bacteria (Escherichia coli ATCC ® 25922 and P. aeruginosa ATCC ® 27853), even at the highest concentrations tested (32 μg/mL, maximum solubility for the compounds). The antibacterial screening results are presented in Table 1. However, preliminary tests showed that adding EDTA to compounds [21] may induce activity against Gram-negative bacteria (unpublished results), through two possible mechanisms: fluidizing the lipid membrane [21] to facilitate passage of the derivatives through the membrane; or enabling the compound to act as a siderophore, in which the compound takes advantage of the high affinity of the iron transport system to facilitate entry into the bacterium [22]. This result suggests that it is possible to achieve activity against Gram-negative bacteria using a strategy that enables penetration of the bacterial membrane.

Structure-Activity Relationship
Core 1 showed no antibacterial activity, but addition of the thiophenol ring resulted in measurable activity (compound 2). This observation suggests that the aromatic system is essential to activity. Substituents at the 2 -, 3 -, and 4 -positions of the thiophenol ring gave rise to the 2 -, 3 -, and 4 -series, respectively. The 2 -series was comprised of compounds 3 through 7. Using the activity of compound 2 as a reference, compounds 4 and 7 exhibited the highest antibiotic activity within this series and possessed the bulkiest substituents in the series, the methoxy group and the bromine atom, with MR values of 7.24 and 9.06 cm 3 /mol, respectively. However, the presence of a methyl group in compound 3 was associated with reduced activity, resulting in MIC values of 32 µg/mL for MRSA and MSSA and >32 µg/mL for E. faecalis. Finally, fluorine and chlorine substituents in the ortho position, which produced compounds 5 and 6, respectively, showed no antibacterial activity. It is interesting to note the steric effect of the substituents at the C-2 position of the thiophenol ring; apparently, the atomic volume forces the structure to assume a conformation with a semi-perpendicular dihedral angle that may reduce the degrees of freedom of the structure. This observation suggests that bulky groups at C-2 favor antibacterial activity.
Compounds with meta substitutions, the 3 -series (compounds 8 through 12), showed MIC values of 2 to 32 µg/mL. Within this series, it was not possible to obtain a correlation that explained the results of biological activity obtained according to the LogP, E 1/2 , or MR molecular descriptors. However, the methyl, methoxy, and fluorine substituents (compounds 8, 9, and 10, respectively) were observed to have the best profiles in Gram-positive bacteria. On the other hand, interestingly, the halogenated compounds (11 and 12) were more potent against resistant than sensitive strains, because these compounds showed MIC values of 2 µg/mL MRSA strains, whereas potency against MSSA strains was lower, at 32 µg/mL. The 4 -series compounds (13 through 17) showed MIC values ranging from 4 to 16 µg/mL. compound 16 showed the highest potency. Considering the reported relationships between the quinonic structures, their redox capacity, and their biological activity [24,25], we tackled the study of the 4 -series compounds, considering that the thiophenol substituents could modulate antibacterial activity through control of the descriptor E 1/2 .
The relationship established by Hammett between the type of substituent in the para position and reactivity is well known [26]. This analysis can be applied to the 4 -series, in that whether the substituent in position 4 is an electron donor or acceptor will affect the quinonic nucleus through the sulfur atom, modifying the redox potential of the compounds. This effect requires that the aromatic systems, the thiophenolic ring and the quinonic nucleus, are in the same plane to maximize conjugation of the two systems, which can be extrapolated according to the value of the dihedral angle between the two aromatic systems. In order to determine the dihedral angle, compound 16 was crystallized and resolved using X-ray diffraction to produce a three-dimensional image of the structure. In this analysis, the most active compound of the 2 -series (compound 7) was included to determine whether the insertion of a bulky substituent in position 2 (bromine atom) significantly influences the dihedral angle. Images of the molecular structure showed that the dihedral angle between the aromatic ring and the quinone core was nearly perpendicular, with values of 81.6 and 76.5 degrees for compounds 7 and 16, respectively ( Figure 2).  These results demonstrate than the molecules do not present a dihedral angle sufficient to consider significant conjugation between the aromatic systems; therefore, the electronic effect of the substituent on the half-wave potential must be principally inductive. This observation is concordant with the narrow range of E1/2 values obtained for the compounds (−0.623 to −0.374 mV), suggesting that the inductive effect weakly affected the redox potential of the quinones. Interestingly, the bulky substituent in the ortho or para position did not significantly alter the dihedral angle, because the presence of the bromine atom in position 2′ increased the angle by 5 degrees as compared to molecules with a chloride atom in position These results demonstrate than the molecules do not present a dihedral angle sufficient to consider significant conjugation between the aromatic systems; therefore, the electronic effect of the substituent on the half-wave potential must be principally inductive. This observation is concordant with the narrow range of E 1/2 values obtained for the compounds (−0.623 to −0.374 mV), suggesting that the inductive effect weakly affected the redox potential of the quinones. Interestingly, the bulky substituent in the ortho or para position did not significantly alter the dihedral angle, because the presence of the bromine atom in position 2 increased the angle by 5 degrees as compared to molecules with a chloride atom in position 4 . This analysis for the 4 series shows that the E 1/2 parameter does not present a clear correlation with antibacterial activity. With respect to the lipophilicity parameter, no correlation was observed with activity in the 2 series, since the compound with the lowest (compound 4) and highest (compound 7) logP showed similar activities in the series. For the 3 series, despite the variation in lipophilicity, a narrow range of MIC values (2 to 8 ug/mL) was observed. Of the MSSA, the most lipophilic compounds of the 3 series had the lowest activity. For the 4 series, a linear correlation between logP and MIC was observed, where the compounds with the highest lipophilicity had the highest activities of the series.
In summary, the phenyl ring is essential for activity, the increased size of substituents in the ortho position improve antibacterial activity, and for the meta position, the physicochemical characteristics of the substituents do not show a correlation with antibacterial activity. In the para position, a lipophilic substituent improves antibacterial activity.

Antibacterial Activity of Compounds 7 and 16 Against Clinical MDR Isolates
To assess the clinical potential of the most promising compounds, we evaluated their antibacterial activity against heterogeneous populations of clinical isolates as well as their toxicity against human and animal cells. Compounds 7 and 16 were selected for this analysis, as these two compounds showed the greatest antibacterial activity.
The MIC 50/90 and MBC 50/90 against clinical isolates of MRSA and vancomycin-resistant E. faecium (VREF), as well as the geometric mean (MG) of the MIC parameters, were determined for compounds 7 and 16. The MIC 50/90 values reflect the effectiveness of antibacterial agents against a heterogeneous bacterial population. Vancomycin (VAN) and gentamicin (GEN) were used as quality controls; if the MIC values for these reference drugs against each bacterial strain were equal to the values reported by the Instituto de Salud Pública de Chile (ISP), the trials were considered valid. The maximum concentration tested was 32 µg/mL, as this value represents the solubility limit of the compounds. The results obtained for compounds 7, 16, and VAN against MRSA and VREF are summarized in Tables 2 and 3, respectively, and the histograms are presented in Figure 3. For compound 7, the MIC 50/90 was 2/2 µg/mL, and the MBC 50/90 was 4/4 µg/mL in 32 S. aureus isolates. For compound 16, in contrast, the MIC 50/90 was 2/4 µg/mL, and the MBC 50/90 was 2/4 µg/mL in 29 S. aureus isolates. For VAN   [a] isolates with MIC > 32 were not considered in the analysis; [b] MIC Minimum Inhibitory Concentration (μg/mL); [c] geometric mean; [d] : Minimum Bactericidal Concentration (μg/mL); ND: Not determined. In triplicate and n = 5.

Antibacterial Activity Against Heterogeneous Populations of Clinical Isolates
The bacterial population used to calculate the MIC90 values had a complex susceptibility profile, as most of the isolates tested were resistant to three or more classes of drugs. However, both compounds had MIC90 values near those of VAN against MRSA (MIC90 = 1 μg/mL, p > 0.001). Moreover, the two compounds had significantly lower MIC values than VAN against VREF isolates (64-256 μg/mL, p > 0.001). Compounds 7 and 16 were 128-fold and 64-fold more active than VAN, respectively, against the various clinical isolates of VREF. The MIC90 values for compounds 7 and 16 are similar to those reported for linezolid (2-4 μg/mL) against MRSA [27][28][29][30][31], showing that these molecules may offer potential therapeutic alternatives to VAN for the treatment of skin infections caused by MRSA [32]. Linezolid has been found to have MIC90 values of 2-64 μg/mL against Enterococcus spp. [27,29,33], indicating that linezolid is a therapeutic option for treating urinary tract infections caused by VREF. In addition, compounds 7 and 16 showed activity similar to or greater than VAN and linezolid against MRSA and multidrug-resistant E. faecium isolates. Furthermore, compounds 7 and 16 were found to have MBC values equal to or up to two times greater than the MIC values against all strains tested, including MRSA (32 isolates) and VREF (44 isolates). An MBC/MIC ratio of 2 or lower indicates that a compound has a bactericidal mode of action, according to Craig et al. [34], suggesting that these compounds were successful in killing the clinical isolates of MRSA and VREF.

Antibacterial Activity Against Heterogeneous Populations of Clinical Isolates
The bacterial population used to calculate the MIC 90 values had a complex susceptibility profile, as most of the isolates tested were resistant to three or more classes of drugs. However, both compounds had MIC 90 values near those of VAN against MRSA (MIC 90 = 1 µg/mL, p > 0.001). Moreover, the two compounds had significantly lower MIC values than VAN against VREF isolates (64-256 µg/mL, p > 0.001). Compounds 7 and 16 were 128-fold and 64-fold more active than VAN, respectively, against the various clinical isolates of VREF. The MIC 90 values for compounds 7 and 16 are similar to those reported for linezolid (2-4 µg/mL) against MRSA [27][28][29][30][31], showing that these molecules may offer potential therapeutic alternatives to VAN for the treatment of skin infections caused by MRSA [32]. Linezolid has been found to have MIC 90 values of 2-64 µg/mL against Enterococcus spp. [27,29,33], indicating that linezolid is a therapeutic option for treating urinary tract infections caused by VREF. In addition, compounds 7 and 16 showed activity similar to or greater than VAN and linezolid against MRSA and multidrug-resistant E. faecium isolates. Furthermore, compounds 7 and 16 were found to have MBC values equal to or up to two times greater than the MIC values against all strains tested, including MRSA (32 isolates) and VREF (44 isolates). An MBC/MIC ratio of 2 or lower indicates that a compound has a bactericidal mode of action, according to Craig et al. [34], suggesting that these compounds were successful in killing the clinical isolates of MRSA and VREF.

Cytotoxicity of Compounds 7 and 16
To test the safety of compounds 7 and 16 in mammalian cells, cytotoxicity studies were performed using the HeLa, HTC-116, SH-SY5Y, and Vero cell lines. A colorimetric assay was performed to estimate the half-maximal inhibitory concentration (IC 50 ) values, which represent the concentration of a drug that is required for 50% inhibition in vitro after 24 h of continuous exposure to the compound. Four serial dilutions (from 0.02 to 20 µg/mL) for each sample were evaluated in triplicate in three reproducible assays, and VAN and GEN were used as reference drugs. The results are shown in Figure 4.  It was not possible to determine the IC50 values because the viability percentages observed were greater than 75% at all concentrations studied. No significant differences between compounds 7 or 16 (p > 0.001) and the positive control (p > 0.001) or the reference drugs (p > 0.001) were observed.
The toxicity results for mammalian cells, including human (HeLa, HTC-116, and SHSY-5Y) and animal (Vero) cells, showed no significant differences between either of the two compounds and the positive control (p > 0.05), indicating that the compounds did not affect the viability of the cells after 24 h of exposure at any of the concentrations studied (0.002-20 μg/mL). Moreover, there were no significant differences in toxicity between the two novel compounds and the reference antibacterial agents (VAN and GEN) at any of the concentrations tested (p > 0.05). Due to the low solubility of the compounds, it was not possible to determine their IC50 in cell culture. At the maximum concentration tested, 70% cell viability was observed, a percentage that is insufficient for calculation of the IC50.
Finally, there was a wide gap between the antibiotic and toxic concentrations of the compounds. For example, it would be necessary to increase the concentration of compounds 7 and 16, which showed MIC values of 2 and 4 μg/mL against MRSA, respectively, by a factor of 5 to 10 before the viability of mammalian cells (HeLa and Vero) would be affected. It was not possible to determine the IC 50 values because the viability percentages observed were greater than 75% at all concentrations studied. No significant differences between compounds 7 or 16 (p > 0.001) and the positive control (p > 0.001) or the reference drugs (p > 0.001) were observed.

Materials and Methods
The toxicity results for mammalian cells, including human (HeLa, HTC-116, and SHSY-5Y) and animal (Vero) cells, showed no significant differences between either of the two compounds and the positive control (p > 0.05), indicating that the compounds did not affect the viability of the cells after 24 h of exposure at any of the concentrations studied (0.002-20 µg/mL). Moreover, there were no significant differences in toxicity between the two novel compounds and the reference antibacterial agents (VAN and GEN) at any of the concentrations tested (p > 0.05). Due to the low solubility of the compounds, it was not possible to determine their IC 50 in cell culture. At the maximum concentration tested, 70% cell viability was observed, a percentage that is insufficient for calculation of the IC 50 . Finally, there was a wide gap between the antibiotic and toxic concentrations of the compounds. For example, it would be necessary to increase the concentration of compounds 7 and 16, which showed MIC values of 2 and 4 µg/mL against MRSA, respectively, by a factor of 5 to 10 before the viability of mammalian cells (HeLa and Vero) would be affected.

General Procedure for the Synthesis of Compounds 2-17
A solution of 1 (150 mg, 0.4909 mmol) and CeCl3.7H2O (5% mmol respect to 1) in a mix of ethanol: oromethane = 1:1 (10 mL), was added dropwise slowly a solution of benzenethiol derivate (0.5 .) in ethanol: dichloromethane = 1:1 (30 mL). The reaction mixture was stirred at room temperature 6 h. The progress of the reaction was followed by thin-layer chromatography (TLC). The mixture oncentrated and the crude of reaction was purified using 65 g of silica gel (70-230 mesh) and a mix chloromethane, light petroleum and ethyl acetate than eluent in determinate proportions. The ting solution was concentrated to dryness under reduced pressure.

General Procedure for the Synthesis of Compounds 2-17
A solution of 1 (150 mg, 0.4909 mmol) and CeCl 3 .7H 2 O (5% mmol respect to 1) in a mix of ethanol: dichloromethane = 1:1 (10 mL), was added dropwise slowly a solution of benzenethiol derivate (0.5 equiv.) in ethanol: dichloromethane = 1:1 (30 mL). The reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was followed by thin-layer chromatography (TLC). The mixture was concentrated and the crude of reaction was purified using 65 g of silica gel (70-230 mesh) and a mix of dichloromethane, light petroleum and ethyl acetate than eluent in determinate proportions. The resulting solution was concentrated to dryness under reduced pressure.

General Procedure for the Synthesis of Compounds 2-17
A solution of 1 (150 mg, 0.4909 mmol) and CeCl3.7H2O (5% mmol respect to 1) in a mix of ethanol: dichloromethane = 1:1 (10 mL), was added dropwise slowly a solution of benzenethiol derivate (0.5 equiv.) in ethanol: dichloromethane = 1:1 (30 mL). The reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was followed by thin-layer chromatography (TLC). The mixture was concentrated and the crude of reaction was purified using 65 g of silica gel (70-230 mesh) and a mix of dichloromethane, light petroleum and ethyl acetate than eluent in determinate proportions. The resulting solution was concentrated to dryness under reduced pressure. (2) (Kenilworth, NJ, USA). All solvents were commercially available and of reagent grade and were used without further purification. Melting points (mp) were determined on a SMP3 apparatus (Stuart Scientific, Staffordshire, United Kingdom) and were uncorrected. 1 H-NMR spectra (400 MHz) were recorded on an AM-400 instrument (Bruker, Billerica, MA, USA) in deuterochloroform (CDCl3). 13 C-NMR spectra were obtained in CDCl3 at 100 MHz. Peak assignment was confirmed by correlation with chemical structures in 2D experiments (HMBC, HSQC) performed by Valderrama et al. [35][36][37][38][39]. The assignments of chemical shifts are expressed in ppm downfield relative to tetramethylsilane (TMS, δ scale), and the coupling constants (J) are reported in Hertz. IR spectra were recorded on a Bruker Vector 22-FT spectrophotometer using KBr discs, and wavenumbers are reported in cm −1 . HRMS were obtained on a model MAT 95XP spectrometer (Thermo Finnigan, Barkhausenstr, Germany), Silica gel (70-230 and 230-400 mesh) and TLC aluminum foil 60F254 Merck® were used for preparative column chromatography and analytical TLC, respectively.

General Procedure for the Synthesis of Compounds 2-17
A solution of 1 (150 mg, 0.4909 mmol) and CeCl3.7H2O (5% mmol respect to 1) in a mix of ethanol: dichloromethane = 1:1 (10 mL), was added dropwise slowly a solution of benzenethiol derivate (0.5 equiv.) in ethanol: dichloromethane = 1:1 (30 mL). The reaction mixture was stirred at room temperature for 16 h. The progress of the reaction was followed by thin-layer chromatography (TLC). The mixture was concentrated and the crude of reaction was purified using 65 g of silica gel (70-230 mesh) and a mix of dichloromethane, light petroleum and ethyl acetate than eluent in determinate proportions. The resulting solution was concentrated to dryness under reduced pressure. 6-Ethyl-8-(phenylthio)-2,4-dimethylpyrimido [4,5-c] , and benzenethiol, from Merck ® (Kenilworth, NJ, USA). All solvents were commercially available and of reagent grade and were used without further purification. Melting points (mp) were determined on a SMP3 apparatus (Stuart Scientific, Staffordshire, United Kingdom) and were uncorrected. 1 H-NMR spectra (400 MHz) were recorded on an AM-400 instrument (Bruker, Billerica, MA, USA) in deuterochloroform (CDCl3). 13 C-NMR spectra were obtained in CDCl3 at 100 MHz. Peak assignment was confirmed by correlation with chemical structures in 2D experiments (HMBC, HSQC) performed by Valderrama et al. [35][36][37][38][39]. The assignments of chemical shifts are expressed in ppm downfield relative to tetramethylsilane (TMS, δ scale), and the coupling constants (J) are reported in Hertz. IR spectra were recorded on a Bruker Vector 22-FT spectrophotometer using KBr discs, and wavenumbers are reported in cm −1 . HRMS were obtained on a model MAT 95XP spectrometer (Thermo Finnigan, Barkhausenstr, Germany), Silica gel (70-230 and 230-400 mesh) and TLC aluminum foil 60F254 Merck® were used for preparative column chromatography and analytical TLC, respectively.

Preparation of Single Crystals
Single crystals were grown by solvent evaporation at room temperature from the products of synthesis. Crystals suitable for X-ray diffraction studies were obtained from crystallization in saturated solutions: tetrahydropyran for 7 and benzene for 16.

Single Crystal X-ray Diffraction
Measured crystals were prepared under inert conditions and immersed in perfluoropolyether as a protective oil for manipulation. Suitable crystals were mounted on MiTeGen MicromountsTM, and these samples were used for data collection. Data were collected with a D8 Venture diffractometer CuKα, 298 K (Bruker, Karlsruhe, Germany). The data were processed with the APEX3 program [40] and corrected for absorption using SADABS [41]. The structures were resolved using direct methods [42], which revealed the position of all nonhydrogen atoms (Figures S1 and S2). These atoms were refined on F2 by a full-matrix least-squares procedure using anisotropic displacement parameters. All hydrogen atoms were located in difference Fourier maps and were included as fixed contributions riding on attached atoms with isotropic thermal displacement parameters 1.2 (C-H) or 1.5 (methyl) times those of the respective atom. CCDC 1573897 (compound 7) and 1573896 (compound 16) contain the crystallographic data listed in Table S2. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre.

Bacterial Strains
As an initial screen for antibacterial activity, the compounds were tested against the following strains: methicillin-resistant Staphylococcus aureus (MRSA) ATCC ® 43300, methicillin-susceptible Staphylococcus aureus (MSSA) ATCC ® 29213, Enterococcus faecalis ATCC ® 29212, Escherichia coli ATCC ® 25922, and Pseudomonas aeruginosa ATCC ® 27853. The ISP provided the clinical isolates of MRSA [31] and VREF [32] used to calculate the MIC and MBC values of the most potent compounds. MRSA and VREF isolates that were resistant to at least three additional classes of antimicrobial agents were defined as multidrug resistant (MDR) [43]. All isolates were collected in 2014 and were obtained from various Chilean hospitals throughout the country (Table S1, Supplementary Material).

Evaluation of Antibacterial Activity
Minimal Inhibitory Concentration Determination MIC values were determined using a broth microdilution method, according to recommendations of the Clinical and Laboratory Standards Institute (CLSI) [44]. VAN and GEN were also tested against the strains and the results compared to the MIC ranges reported by the CLSI, as a quality control measure [23]. All compounds tested were dissolved in dimethyl sulfoxide (DMSO), to levels not exceeding 1% per well. MIC 50 and MIC 90 values were then calculated for the compounds observed to be the most active against the clinical isolates studied were also determined; MIC 50 and MIC 90 are the concentrations that inhibit 50% and 90% of the tested isolates, respectively. All assays were performed in triplicate and with n = 5.

Minimal bactericidal concentration determination
MBC values were then calculated for the compounds observed to be the most active against clinical isolates, following the recommendations of Pearson et al. [45], using an inoculum of 5 × 10 5 CFU/mL. All wells with no visible growth observed in the microdilution assay were subcultured, extracting a 10-µL volume. Finally, MBC was determined based on the corresponding subcultured dilution plate in which the growth of microorganisms was less than 0.01% of the initial inoculum of 5 × 10 5 CFU/mL. Compounds were classified as bactericides if the MBC/MIC ratio was equal to or less than 2 and as bacteriostatic if the ratio was greater than 2, according to Craig et al. [46] and Taylor et al. [47]. All assays were performed in triplicate and with n = 5.

Evaluation of Cellular Toxicity
Cytotoxicity assays were performed using the MTS reduction method, as described previously [48]. Briefly, cancer cell lines were plated in a flat-bottom 96-well plate at 40,000 cells/mL. The cells were then incubated with test agents and VAN at escalating doses, ranging from 0 to 20 µg/mL, in triplicate in 200 µL of RPMI 1640 supplemented culture medium at 37 • C for 24 h. Five microliters of MTS was added to achieve a final concentration of 0.5 mg/mL and incubated at 37 • C for 2 h. Finally, formazan formation was measured in a multi-well reader at 540 nm (Stat Fax 4200, Awareness Technology, Inc., Palm City, FL, USA). The compounds were dissolved in DMSO, at levels not exceeding 1% per well. All assays were performed in triplicate and with n = 5. As a positive control for growth, wells containing only RPMI 140 medium were used. As a negative control of growth, H 2 O 2 was used. As an additional negative control for growth, H 2 O + 1% DMSO was used. VAN and GEN were used as drug references.

Electrochemical Measuring
Cyclic voltammetry: Half-wave potentials (E 1/2 ) were determined using a CHI 650 potentiostat (CH Instruments, Inc., 3700 Tennison Hill Drive Austin, TX, USA). Three-electrode cells with Ag 0 /AgCl or platinum wires were used as reference and auxiliary electrodes, respectively. The working electrode was prepared by polishing a glassy carbon electrode with 0.3 µm and 0.05 µm alumina and then washing with abundant deionized water. The compounds were dissolved to a concentration of 0.1 M with acetonitrile as a solvent and tetrabutylammonium perchlorate as a support electrolyte.

Statistical Analysis
The data were analyzed with one-way ANOVA and t-tests, with the criterion for statistical significance set at p < 0.05, using the GraphPad Prism 5.03 program (GraphPad Software, Inc., San Diego, California, USA, www.graphpad.com).

Conclusions
We synthesized 17 novel quinone compounds by employing a simple, fast, and economical two-step method with a yield of 47-88% yield. Antibacterial screening showed the compounds to have activity against Gram-positive MRSA, MSSA, and E. faecalis strains but not against Gram-negative E.coli and P. aeruginosa.
Structural analysis showed that the presence of a thiophenolic ring is essential for activity. On the other hand, a study of the LogP, E 1/2 , and MR parameters showed that the addition of bulky substituents in the ortho position or the addition of lipophilic substituents in the para position improves the antibacterial activity. The results suggest that the antibacterial activity would be a function of more complex parameters that require extensive analysis.
Compounds 7 and 16 were 128-and 64-fold more active against clinical isolates of VREF, respectively, than the drug VAN and did not affect the viability of HeLa, HTC-116, SHSY-5Y, or Vero cells in toxicity assays, demonstrating selectivity for prokaryotic cells. From a drug development point of view, the results of the new scaffold described herein support continuation of preclinical development to guide the synthesis of more potent agents to provide therapeutic options against infectious diseases provoked by Gram-positive MDR strains.