Antistaphylococcal Activities and ADME-Related Properties of Chlorinated Arylcarbamoylnaphthalenylcarbamates

Pattern 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide and the thirteen original carbamates derived from it were prepared and characterized. All the compounds were tested against Staphylococcus aureus ATCC 29213 as a reference and quality control strain and in addition against three clinical isolates of methicillin-resistant S. aureus (MRSA). Moreover, the compounds were evaluated against Enterococcus faecalis ATCC 29212, and preliminary in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line (THP-1). The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. While pattern anilide had no antibacterial activity, the prepared carbamates demonstrated high antistaphylococcal activity comparable to the used standards (ampicillin and ciprofloxacin), which unfortunately were ineffective against E. feacalis. 2-[(2,4,5-Trichlorophenyl)carba- moyl]naphthalen-1-yl ethylcarbamate (2) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl butylcarbamate (4) expressed the nanomolar minimum inhibitory concentrations (MICs 0.018–0.064 μM) against S. aureus and at least two other MRSA isolates. Microbicidal effects based on the minimum bactericidal concentrations (MBCs) against all the tested staphylococci were found for nine carbamates, while 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl heptylcarbamate (7) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl (4-phenylbutyl)carbamate (14) demonstrated MBCs in the range of 0.124–0.461 μM. The selectivity index (SI) for most investigated carbamates was >20 and for some derivatives even >100. The performed tests did not show an effect on the damage to the bacterial membrane, while the compounds were able to inhibit the respiratory chain of S. aureus.


Introduction
Staphylococcus aureus, discovered by Scottish surgeon A. Ogston in 1880 and named by the German bacteriologist F. Rosenbach in 1884, remains one of the most common and re-

Chemistry
The synthesis of the target carbamates was performed in two steps (Scheme 2). First, 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide (1) was prepared by the microwaveassisted method using phosphorus trichloride in dry chlorobenzene from 1-hydroxynaphthalene-2-carboxylic acid and 2,4,5-trichloroaniline [31]. In the second step, the triethylamine-activated phenolic group was reacted with the appropriate isocyanate in acetonitrile to provide a series of final 2-[(2,4,5-trichlorophenyl)carbamoyl]-naphthalen-1-yl alkyl-, cycloalkyl-, and arylalkyl-carbamates 2-14, see Table 1. The compounds were studied in detail for their physicochemical properties, as they meet the basic drug-likeness requirements, represented in particular by the Lipinski's Rule of Five (Ro5) [32]. Lipophilicity was measured by reversed-phase high performance liquid chromatography (RP-HPLC) under isocratic conditions with methanol as the organic modifier in the aqueous mobile phase and the logarithm of the capacity factor k was calculated, which is used as the lipophilicity index converted to the log P scale [33,34]. In addition to the log k values, the logarithm of the distribution coefficient DpH at pH 6.5 and 7.4 was determined to clarify the behavior of the compounds under physiological conditions and possible ionization [33,35]. Instead of water, a buffer of suitable pH was used as Scheme 1. Design of the investigated carbamates based on structural analogy/bioisosterism to salicylanilide derivatives.
Scheme 1. Design of the investigated carbamates based on structural analogy/bioisosterism to salicylanilide derivatives.

Chemistry
The synthesis of the target carbamates was performed in two steps (Scheme 2). First, 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide (1) was prepared by the microwaveassisted method using phosphorus trichloride in dry chlorobenzene from 1-hydroxynaphthalene-2-carboxylic acid and 2,4,5-trichloroaniline [31]. In the second step, the triethylamine-activated phenolic group was reacted with the appropriate isocyanate in acetonitrile to provide a series of final 2-[(2,4,5-trichlorophenyl)carbamoyl]-naphthalen-1-yl alkyl-, cycloalkyl-, and arylalkyl-carbamates 2-14, see Table 1. The compounds were studied in detail for their physicochemical properties, as they meet the basic drug-likeness requirements, represented in particular by the Lipinski's Rule of Five (Ro5) [32]. Lipophilicity was measured by reversed-phase high performance liquid chromatography (RP-HPLC) under isocratic conditions with methanol as the organic modifier in the aqueous mobile phase and the logarithm of the capacity factor k was calculated, which is used as the lipophilicity index converted to the log P scale [33,34]. In addition to the log k values, the logarithm of the distribution coefficient DpH at pH 6.5 and 7.4 was determined to clarify the behavior of the compounds under physiological conditions and possible ionization [33,35]. Instead of water, a buffer of suitable pH was used as The compounds were studied in detail for their physicochemical properties, as they meet the basic drug-likeness requirements, represented in particular by the Lipinski's Rule of Five (Ro5) [32]. Lipophilicity was measured by reversed-phase high performance liquid chromatography (RP-HPLC) under isocratic conditions with methanol as the organic modifier in the aqueous mobile phase and the logarithm of the capacity factor k was calculated, which is used as the lipophilicity index converted to the log P scale [33,34]. In addition to the log k values, the logarithm of the distribution coefficient D pH at pH 6.5 and 7.4 was determined to clarify the behavior of the compounds under physiological conditions and possible ionization [33,35]. Instead of water, a buffer of suitable pH was used as part of the mobile phase. In addition to the above-mentioned experimental methods, commercially available ChemBioDraw Ultra 13.0 and ACD/Percepta ver. 2012 programs were used to calculate the lipophilicity values of all the prepared compounds. All the results are shown in Table 1. In addition to lipophilicity, other parameters discussed in Ro5, such as molecular weight, number of H-bond donors/acceptors, as well as parameters not reported in Ro5, but characterizing the physicochemical properties of the prepared molecules, such as number of rotatable bonds, were predicted using ACD/Percepta. Moreover, for each R substituent, bulkiness expressed as molar volume (MV [cm 3 ]), a parameter describing the tail length/branching of each carbamates, was calculated. All these parameters are listed in Table 1. part of the mobile phase. In addition to the above-mentioned experimental methods, commercially available ChemBioDraw Ultra 13.0 and ACD/Percepta ver. 2012 programs were used to calculate the lipophilicity values of all the prepared compounds. All the results are shown in Table 1. In addition to lipophilicity, other parameters discussed in Ro5, such as molecular weight, number of H-bond donors/acceptors, as well as parameters not reported in Ro5, but characterizing the physicochemical properties of the prepared molecules, such as number of rotatable bonds, were predicted using ACD/Percepta. Moreover, for each R substituent, bulkiness expressed as molar volume (MV [cm 3 ]), a parameter describing the tail length/branching of each carbamates, was calculated. All these parameters are listed in Table 1. From the experimental lipophilicity results, it is evident that the starting anilide 1 has the lowest values of log k and log D. (Note that this fact was correctly calculated only by log P from ACD/Percepta. ChemDrawUltra predicted ethyl carbamate 2 to be the least lipophilic.) Within the series of linear tail derivatives 2-8, lipophilicity increases linearly from the ethyl (ca. 0.83) to octyl (ca. 1.87), which shows the highest lipophilicity of all carbamates. Within cyclic derivatives 10-13, lipophilicity increases from cyclopentyl (ca. 1.15) to cycloheptyl (ca. 1.45). Logically, the phenylethyl derivative has a lower lipophilicity than the phenylbutyl derivative (14, ca. 1.25 and 15 ca. 1.52, respectively). The lipophilicity of the isopropyl derivative 9 is slightly lower (insignificantly different) from the propyl derivative 3. The lipophilicity of cyclic derivatives 10-13 ranges from the level of aliphatic derivatives from butyl to hexyl carbamates 4-6. The lipophilicity of the phenylalkyl carbamates 13 and 14 is at the level of pentyl/hexyl 5/6 and cyclohexyl/cycloheptyl 11/12 derivatives, respectively. It is important to note that all experimental lipophilicity values correlated with each other in the interval of the correlation coefficients r from 0.9908 to 0.9974 (n = 13), as shown in the graphs of Figure S1. Deviations between the log k/log D values were most evident in the parent anilide 1, where with increasing pH, lipophilicity decreased by almost 20% (log k = 0.6600, log D 7.4 = 0.5384), which may indicate the ability of the phenolic hydroxyl to be protonated in the basic pH medium. No significant changes in the experimental lipophilicity values were found for any of the carbamates 2-14.
The correlation between experimental and predicted lipophilicity values in the carbamate series 2-14 (n = 13) was the most pronounced for Clog P (ChemDrawUltra, ranged from 0.9826 to 0.9900), followed by log P (ACD/Percepta, ranged from 0.9585 to 0.9631), and the worst correlation was observed for log P (ChemDrawUltra, ranged from 0.9426 to 0.9505). The graphs are shown in Figures S2-S4. In general, the log k values correlated best with the predicted data, and log D 7.4 the worst; the best and worst correlations are shown in Figure 1. The largest deviations were found for phenylalkyl derivatives 13 and 14 (for log P values obtained by ChemDrawUltra), smaller for cycloheptyl (for log P values obtained by ACD/Percepta). alkyl carbamates 13 and 14 is at the level of pentyl/hexyl 5/6 and cyclohexyl/cycloheptyl 11/12 derivatives, respectively. It is important to note that all experimental lipophilicity values correlated with each other in the interval of the correlation coefficients r from 0.9908 to 0.9974 (n = 13), as shown in the graphs of Figure S1. Deviations between the log k/log D values were most evident in the parent anilide 1, where with increasing pH, lipophilicity decreased by almost 20% (log k = 0.6600, log D7.4 = 0.5384), which may indicate the ability of the phenolic hydroxyl to be protonated in the basic pH medium. No significant changes in the experimental lipophilicity values were found for any of the carbamates 2-14. The correlation between experimental and predicted lipophilicity values in the carbamate series 2-14 (n =13) was the most pronounced for Clog P (ChemDrawUltra, ranged from 0.9826 to 0.9900), followed by log P (ACD/Percepta, ranged from 0.9585 to 0.9631), and the worst correlation was observed for log P (ChemDrawUltra, ranged from 0.9426 to 0.9505). The graphs are shown in Figures S2-S4. In general, the log k values correlated best with the predicted data, and log D7.4 the worst; the best and worst correlations are shown in Figure 1. The largest deviations were found for phenylalkyl derivatives 13 and 14 (for log P values obtained by ChemDrawUltra), smaller for cycloheptyl (for log P values obtained by ACD/Percepta). Based on the lipophilicity values given in Table 1, it can be stated that even the starting anilide no longer meets the Ro5 recommendation of log P <5. On the other hand, there is a chance that the compounds will penetrate better through the bacterial wall. Likewise, heptyl, octyl, cycloheptyl, and both fenylalkyl derivatives have slightly higher molecular weights than recommended. In the other parameters, the compounds meet the Ro5 recommendations. For a more detailed characterization of the carbamate tails, Table 1 shows their bulkiness expressed as molar volumes (MV (cm 3 )) of the individual carbamate substituents R. Logically, the ethyl chain (compound 2) is the least bulky, while octyl and phenylbutyl substituents (compounds 8, 14) are the bulkiest. Previous studies that focused on the physicochemical and biological properties of carbamates (or, in general, agents with a polar head and a lipophilic tail) have found that the ability of these molecules to disrupt the integrity of biological membranes is extremely important for any of their biological activities [14,19,20,[36][37][38][39]. The selection of a suitable chain length is extremely important and difficult for the ability of the agent to act as a surfactant, because tail length subsequently strongly affects the other related physicochemical properties of the compound. Thus, the investigated series of carbamates is characterized by a wide structural spectrum of tails. In general, the longer the lipophilic tail, the better the membrane-damaging properties of the compound. On the other hand, it is necessary to take into account Based on the lipophilicity values given in Table 1, it can be stated that even the starting anilide no longer meets the Ro5 recommendation of log P < 5. On the other hand, there is a chance that the compounds will penetrate better through the bacterial wall. Likewise, heptyl, octyl, cycloheptyl, and both fenylalkyl derivatives have slightly higher molecular weights than recommended. In the other parameters, the compounds meet the Ro5 recommendations. For a more detailed characterization of the carbamate tails, Table 1 shows their bulkiness expressed as molar volumes (MV (cm 3 )) of the individual carbamate substituents R. Logically, the ethyl chain (compound 2) is the least bulky, while octyl and phenylbutyl substituents (compounds 8, 14) are the bulkiest. Previous studies that focused on the physicochemical and biological properties of carbamates (or, in general, agents with a polar head and a lipophilic tail) have found that the ability of these molecules to disrupt the integrity of biological membranes is extremely important for any of their biological activities [14,19,20,[36][37][38][39]. The selection of a suitable chain length is extremely important and difficult for the ability of the agent to act as a surfactant, because tail length subsequently strongly affects the other related physicochemical properties of the compound. Thus, the investigated series of carbamates is characterized by a wide structural spectrum of tails. In general, the longer the lipophilic tail, the better the membrane-damaging properties of the compound. On the other hand, it is necessary to take into account the so-called cut-off effect, which is a non-linear dependence of biological activity on the molecular parameter of the surfactant, such as the length of the alkyl chain. This means that the activity only increases to a certain tail length and then decreases. The cut-off effect of the membrane active compounds (which penetrate to the membrane and disrupt its architecture) has been well documented [14,19,20,[36][37][38][39][40][41][42][43][44].

In Vitro Antistaphylococcal Activity
All the investigated compounds were screened for in vitro antibacterial activity against the reference and quality control strain Staphylococcus aureus ATCC 29213 and clinical isolates of methicillin-resistant S. aureus (MRSA) 63718, SA 630, and SA 3202 [14]. Activities are expressed as the minimum inhibitory concentrations (MICs), see Table 2. In addition to the MICs, the minimum bactericidal concentrations (MBCs) were also determined. To establish that a compound demonstrates a bactericidal effect against a particular tested strain, it must meet the condition MIC/MBC ≤ 4 [14,45]. MBC values that meet this requirement, i.e., the compound is bactericidal, are indicated in bold in Table 2. Table 2. In vitro antistaphylococcal and anti-enterococcal activities (MIC/MBC (µM)) compared to ampicillin (AMP) and ciprofloxacin (CPX), and the in vitro cell viability assay (IC 50 (µM) ± SD, n = 6) on the human monocytic leukemia cell line (THP-1) compared to camptothecin (CMT).  It can be stated that, except for starting anilide 1, the all the compounds demonstrated high antistaphylococcal activities against both collection S. aureus and MRSA isolates, which significantly exceeded the activities of the clinically used drugs ampicillin and ciprofloxcin. Overall, ethyl and butyl carbamates 2 and 4 showed the lowest MIC (nanomolar) values against S. aureus and at least two other MRSA isolates, and ethyl 2, butyl 4, and phenylethyl 13 derivatives showed the lowest average MICs against all the tested staphylococci. Nevertheless, it can be stated that the MICs against the MRSA isolates were, in fact, comparable with the MIC values observed against methicillin-susceptible S. aureus ATCC 29213; it could be assumed that the presence of the above-mentioned mecA gene [46] did not affect the activity of these compounds. Microbicidal effects meeting the condition MIC/MBC ≤ 4 for all the tested staphylococci were found for propyl 3, pentyl-octyl 5-8, cyclopentyl-cycloheptyl 10-12, and phenylbutyl 14 carbamates, while derivatives 7 and 14 demonstrated MBCs <0.5 µM.
Encouraged by these results, all the compounds were also evaluated on their antienterococcal activity, but with very unsatisfactory results. MIC values against reference strain Enterococcus faecalis ATCC 29212 are given in Table 2. Only ethyl carbamate 2 showed medium activity, heptyl and cyclohexyl carbamates 7 and 11 demonstrated moderate activity, and the rest of the compounds were completely inactive. A comparison of these highly potent antistaphylococcal agents with moderate or no activity against E. feacalis suggests that the mechanism of action against staphylococci may be different from that of enterococci. An explanation for the observed differences in the susceptibility of staphylococci and enterococci to the tested carbamates can be found in the naturally much greater resistance of enterococci to disinfection procedures [47,48]. Recent functional genomic stud-ies [49,50] identified unique properties of enterococci that allow them to survive exposure to antibiotics.
As mentioned above, the MICs of the compounds against staphylococci are similar; thus, it can be speculated concerning the specific activity against Staphylococcus spp. An attempt was made to reveal the mechanism of action of these compounds, and therefore a standard MTT test was performed with the most active compounds. The MTT assay can be used to assess cell growth by measuring respiration. The MTT measured viability of bacterial cells less than 70% after exposure to the MIC values for each tested compound is considered as a positive result of this assay. This low level of cell viability indicates inhibition of cell growth by inhibition of respiration [19,20,51,52]. It can be concluded that none of the evaluated compounds showed a decrease in viability <70% at its MIC value, which suggests that the main mechanism of action is not inhibition of the respiratory chain, although they are able to significantly affect it, compared to, e.g., ciprofloxacin. The lowest multiples of the MIC values by which inhibition of S. aureus ATCC 29213 viability (%) greater than 70% was achieved are given in Table 3. As mentioned above, the bactericidal activity of the carbamates was expected to be associated with their ability to disrupt the bacterial membranes. Thus, an alteration in the membrane permeability of S. aureus ATCC 29123 and MRSA SA 630 was detected by a hydrophobic crystal violet dye assay [53,54]. Clinical isolate MRSA2 was selected as the most resistant (bactericidally least sensitive) to the evaluated carbamates. The bacterial suspensions were treated by compounds 2-4, 7-9, 13, and 14 (4× MIC) for 1 h. The uptake of crystal violet was expressed as a percentage compared to the original crystal violet solution. The results are shown in Figure 2. The test compounds did not affect plasma membrane permeability of either S. aureus or MRSA, because the percentage absorption of crystal violet was comparable to the growth control and disproportionately lower than the 1% Tween 20 solution used as the positive control. Based on these results, it could be assumed that an increase in the membrane permeability was not the main mechanism of action of these compounds.

In Vitro Cell Viability Assay
Preliminary in vitro cytotoxicity screening of the investigated compounds was performed using human monocytic leukemia THP-1 cells, see Table 2. The ability to inhibit the human cancer cell viability (IC 50 ) of the compounds was compared to camptothecin toxicity (0.16 ± 0.07 µM). The cytotoxicity of the parent anilide 1 was found to be insignificant (IC 50 >30 µM), while the ability of cell viability inhibition of carbamates 2-14 was in the range of IC 50 2.61-3.44 µM. However, these values are up to two orders of magnitude higher than their significant antistaphyloccocal concentrations. This fact is also taken into account in the calculated selectivity index (SI), Table 2, where for only three compounds the SI is <20, while for the rest of the agents SI is >20, and for some derivatives even 100 and more. Thus, based on all these observations, it can be concluded that all the tested lution. The results are shown in Figure 2. The test compounds did not affect plasma membrane permeability of either S. aureus or MRSA, because the percentage absorption of crystal violet was comparable to the growth control and disproportionately lower than the 1% Tween 20 solution used as the positive control. Based on these results, it could be assumed that an increase in the membrane permeability was not the main mechanism of action of these compounds.

In Vitro Cell Viability Assay
Preliminary in vitro cytotoxicity screening of the investigated compounds was performed using human monocytic leukemia THP-1 cells, see Table 2. The ability to inhibit the human cancer cell viability (IC50) of the compounds was compared to camptothecin toxicity (0.16 ± 0.07 μM). The cytotoxicity of the parent anilide 1 was found to be insignificant (IC50 >30 μM), while the ability of cell viability inhibition of carbamates 2-14 was in the range of IC50 2.61-3.44 μM. However, these values are up to two orders of magnitude higher than their significant antistaphyloccocal concentrations. This fact is also taken into account in the calculated selectivity index (SI), Table 2, where for only three compounds the SI is <20, while for the rest of the agents SI is >20, and for some derivatives even 100 and more. Thus, based on all these observations, it can be concluded that all the tested derivatives can be considered as in vitro non-toxic compounds for the subsequent design of new therapeutic agents.

Structure-Activity Relationships
As mentioned above, in terms of Ro5, all compounds have a higher lipophilicity than recommended and some derivatives have slightly higher molecular weights. Based on the values from Table 1 and the antistaphylococcal data from Table 2, the dependences of the activities against S. aureus, expressed as log(1/MIC) and log(1/MBC), on lipophilicity (log k) (Figure 3) were created. Both graphs show a clear trend where activity decreases with increasing lipophilicity. Alkyl/phenylalkyl derivatives are clearly separated from cycloalkyl carbamates 10-12 in Figure 3B, which illustrates the dependence of the microbicidal effect on lipophilicity. No significant differences are seen in the graphs of Figure 4, where the dependences of the activities against S. aureus on bulkiness of the carbamate tails, are illustrated. Figures 3B and 4B (the dependences of cidal activities on physicochemical parameters) show a significant bounce of the octyl chain and low activity of octyl carbamate 8 compared to the other derivatives, which is probably related to the above-mentioned cut-off effect [36,37] and "over-bulkiness/over-length" of this tail. It should be noted that the dependences of activity expressed as log(1/MIC) shown in Figures 3A and 4A against all MRSA isolates have only a slightly different course and therefore are not shown. The

Structure-Activity Relationships
As mentioned above, in terms of Ro5, all compounds have a higher lipophilicity than recommended and some derivatives have slightly higher molecular weights. Based on the values from Table 1 and the antistaphylococcal data from Table 2, the dependences of the activities against S. aureus, expressed as log(1/MIC) and log(1/MBC), on lipophilicity (log k) (Figure 3) were created. Both graphs show a clear trend where activity decreases with increasing lipophilicity. Alkyl/phenylalkyl derivatives are clearly separated from cycloalkyl carbamates 10-12 in Figure 3B, which illustrates the dependence of the microbicidal effect on lipophilicity. No significant differences are seen in the graphs of Figure 4, where the dependences of the activities against S. aureus on bulkiness of the carbamate tails, are illustrated. Figures 3B and 4B (the dependences of cidal activities on physicochemical parameters) show a significant bounce of the octyl chain and low activity of octyl carbamate 8 compared to the other derivatives, which is probably related to the above-mentioned cut-off effect [36,37] and "over-bulkiness/over-length" of this tail. It should be noted that the dependences of activity expressed as log(1/MIC) shown in Figures 3A and 4A against all MRSA isolates have only a slightly different course and therefore are not shown. The dependences of the bactericidal activities (log(1/MBC)) against SA 3202 (MRSA3) are also very close to the graphs in Figures 3B and 4B and are not shown.     The dependences of bactericidal activity expressed as log(1/MBC) on the lipophilicity and bulkiness of the carbamate tails against isolates MRSA 63718 and MRSA SA 630 (MRSA1 and MRSA2) have completely different trends than above discussed. As MRSA SA 630 showed the least amount of compounds with bactericidal activity (bold values in Table 2), the dependences are demonstrated on this isolate. Based on these data, it can be stated that the compounds with medium or moderate MIC activity showed mainly bactericidal activity. From the dependence of log(1/MBC) on lipophilicity ( Figure 5A), it can be stated that the activity increases with lipophilicity to the value of log k approx. 1.7 (heptyl derivative 7), then decreases sharply to octyl carbamate 8 with the lowest MBC value. A similar trend can be found in the relationship of bactericidal activity on bulkiness (cidal activity increases up to phenylbutyl derivative 14 with MV ca. 170 cm 3 ), then subsequently decreases sharply to carbamate 8 with the highest bulkiness and the highest lipophilicity, see Figure 5B. Based on the obtained results, it is evident that the bactericidal activity of the studied compounds against MRSA SA 630 and MRSA 63718 is connected to the presence of a longer alkyl chain in the molecule. It can only be speculated whether the different courses of dependences of bactericidal activities against S. aureus ATCC 29213/MRSA SA The dependences of bactericidal activity expressed as log(1/MBC) on the lipophilicity and bulkiness of the carbamate tails against isolates MRSA 63718 and MRSA SA 630 (MRSA1 and MRSA2) have completely different trends than above discussed. As MRSA SA 630 showed the least amount of compounds with bactericidal activity (bold values in Table 2), the dependences are demonstrated on this isolate. Based on these data, it can be stated that the compounds with medium or moderate MIC activity showed mainly bactericidal activity. From the dependence of log(1/MBC) on lipophilicity ( Figure 5A), it can be stated that the activity increases with lipophilicity to the value of log k approx. 1.7 (heptyl derivative 7), then decreases sharply to octyl carbamate 8 with the lowest MBC value. A similar trend can be found in the relationship of bactericidal activity on bulkiness (cidal activity increases up to phenylbutyl derivative 14 with MV ca. 170 cm 3 ), then subsequently decreases sharply to carbamate 8 with the highest bulkiness and the highest lipophilicity, see Figure 5B. Based on the obtained results, it is evident that the bactericidal activity of the studied compounds against MRSA SA 630 and MRSA 63718 is connected to the presence of a longer alkyl chain in the molecule. It can only be speculated whether the different courses of dependences of bactericidal activities against S. aureus ATCC 29213/MRSA SA 3202 (MRSA3) from isolates MRSA1/MRSA2 could be due to their differences in the composition of bacterial membranes [55][56][57][58]. The effect of changing the length of the hydrocarbon tail affecting the extent of antimicrobial activity of the compounds has been discussed (e.g., [36,37,[39][40][41][42][43][44]59,60]). The decrease in biological activity at a higher chain length as a cut-off effect is discussed above. 3202 (MRSA3) from isolates MRSA1/MRSA2 could be due to their differences in the composition of bacterial membranes [55][56][57][58]. The effect of changing the length of the hydrocarbon tail affecting the extent of antimicrobial activity of the compounds has been discussed (e.g., [36,37,[39][40][41][42][43][44]59,60]). The decrease in biological activity at a higher chain length as a cut-off effect is discussed above.

General Methods
All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA). Microwave-assisted reactions were performed

General Methods
All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA). Microwave-assisted reactions were performed using a StartSYNTH microwave lab station (Milestone, Sorisole, Italy). The melting points were determined on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are uncorrected. Infrared (IR) spectra were recorded on an ATR diamond iD7 for Nicolet™ Impact 410 Fourier-transform IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA). The spectra were obtained by the accumulation of 64 scans with a 2 cm −1 resolution in the region of 4000-650 cm −1 . All 1 H-and 13 C-NMR spectra were recorded on an JEOL ECZR 400 MHz NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C, Jeol, Tokyo, Japan) in dimethyl sulfoxide-d 6 (DMSO-d 6 ). 1 H and 13 C chemical shifts (δ) are reported in ppm. High-resolution mass spectra were measured using a high-performance liquid chromatograph Dionex UltiMate ® 3000 (Thermo Scientific, West Palm Beach, FL, USA) coupled with an LTQ Orbitrap XL TM Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo Scientific) equipped with a HESI II (heated electrospray ionization) source in the positive mode.

Lipophilicity Determination by HPLC
A HPLC separation module Agilent 1200 Series (Agilent Technologies, Santa Clara, CA, USA) equipped with a Dual Absorbance Detector (DAD SL G1315C, Agilent Technologies) was used. A chromatographic column Symmetry ® C18 5 µm, 4.6 × 250 mm, Part No. W21751W016 (Waters Corp, Milford, MA, USA) was used. The HPLC separation process was monitored by by ChemStation for LC 3D systems (Agilent Technologies). Isocratic elution by a mixture of MeOH (HPLC grade, 72%) and H 2 O-HPLC Mili-Q grade (28%) as a mobile phase was used for the determination of capacity factor k. Isocratic elution by a mixture of MeOH (HPLC grade, 72%) and acetate buffered saline (pH 7.4 and pH 6.5) (28%) as a mobile phase was used for the determination of distribution coefficient expressed as D 7.4 and D 6.5 . The total flow of the column was 1.0 mL/min, injection 20 µL, column temperature 40 • C, and sample temperature 10 • C. The detection wavelength of 210 nm was chosen. A KI methanolic solution was used for the determination of the dead times (t D ). Retention times (t R ) were measured in minutes. The capacity factors k were calculated according to the formula k = (t R − t D )/t D , where t R is the retention time of the solute, and t D is the dead time obtained using an unretained analyte. The distribution coefficients D pH were calculated according to the formula D pH = (t R − t D )/t D . Each experiment was repeated three times. The log k values of individual compounds are shown in Table 1.

In Vitro Antibacterial Evaluation
In vitro antibacterial activity of the synthesized compounds was evaluated against representatives of multidrug-resistant bacteria, three clinical isolates of methicillin-resistant S. aureus: clinical isolate of animal origin, MRSA 63718 (Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Czech Republic), carrying the mecA gene; and MRSA SA 630 [46] and MRSA SA 3202 [46] (National Institute of Public Health, Prague, Czech Republic), both of human origin. Suspected colonies were confirmed by PCR; a 108bp fragment specific for S. aureus was detected [61]. All isolates were tested for the presence of the mecA gene encoding methicillin resistance [62]. These three clinical isolates were classified as vancomycinsusceptible (but with higher MIC of vancomycin equal to 2 µg/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillin-resistant S. aureus (VS-MRSA) [63]. Vancomycin-susceptible methicillin-susceptible S. aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and quality control strain. Enterococcus faecalis ATCC 29212 was obtained from the American Type Culture Collection and used as the reference and quality control strain.
The minimum inhibitory concentrations (MICs) were evaluated by the microtitration broth method according to the CLSI [64,65] with some modifications. The compounds were dissolved in DMSO (Sigma, St. Louis, MO, USA) to get a concentration 10 µg/mL and diluted in a microtitration plate in an appropriate medium, namely, Cation Adjusted Mueller-Hinton (CaMH, Oxoid, Basingstoke, UK) for staphylococci (S. aureus ATCC 29213, clinical isolates of MRSA 63718, SA 630, and SA 3202) and brain heart infusion (BHI, Oxoid) for E. faecalis ATCC 29212, to reach the final concentration of 256-0.125 µg/mL. The plate was inoculated by the tested microorganisms. The final concentration of bacterial cells was 10 5 for bacteria. Ampicillin (Sigma) was used as reference drugs. A drug-free control and a sterility control were included. The plates were incubated for 24 h at 37 • C for staphylococci and E. faecalis ATCC 29212 (stability evaluation of the carbamates, see Supplementary materials, Table S1). After static incubation in the darkness in an aerobic atmosphere, the MIC was visually evaluated as the lowest concentration of the tested compound, which completely inhibited the growth of the microorganism. The experiments were repeated three times. The results are summarized in Table 2.

Determination of Minimum Bactericidal Concentrations
For the S. aureus and MRSA isolates, the agar aliquot subculture method [66,67] was used as a test for bactericidal agents. After the MIC value determination, the inoculum was transferred to Mueller-Hinton (Oxoid) agar using a multipoint inoculator. The plates were incubated in a thermostat at 37 • C for 24 h. The lowest concentration of test compound at which ≤5 colonies were obtained was then evaluated as MBC, corresponding to a 99.9% decrease in CFU relative to the original inoculum.

MTT Assay
Compounds were prepared as previously stated and diluted in CaMH broth for S. aureus to achieve the desired final concentrations. S. aureus bacterial suspension in sterile distilled water at 0.5 McFarland was diluted 1:3. Inocula were added to each well by multi-inoculator. Diluted mycobacteria in broth free from inhibiting compounds were used as the growth control. All compounds were prepared in duplicate. Plates were incubated at 37 • C for 24 h for S. aureus. After the incubation period, 10% well volume of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (Sigma) was mixed into each well and incubated at 37 • C 1 h for S. aureus. Then 100 µL of 17% sodium dodecyl sulphate in 40% dimethylformamide was added to each well. The plates were read at 570 nm. The absorbance readings from the cells grown in the presence of the tested compounds were compared with uninhibited cell growth to determine the relative percent inhibition. The percent inhibition was determined through the MTT assay. The percent viability is calculated through the comparison of a measured value and that of the uninhibited control: % viability = OD 570E /OD 570P × 100, where OD 570E is the reading from the compound-exposed cells, while OD 570P is the reading from the uninhibited cells (positive control). Cytotoxic potential is determined by a percent viability of <70% [51, 52,68,69]. The results are summarized in Table 3.

Crystal Violet Uptake
The method of crystal violet uptake [53,54] was used to study membrane alteration. A bacterial suspension was cultivated to logarithmic phase in CaMH and harvested at 4500 rpm for 5 min. The cells were washed twice and resuspended in phosphate-buffered saline (PBS) containing 4× MIC of the tested compounds. Tween 20 (1% solution) was used as the positive control. A growth control without antibiotics was included. The tubes were cultivated at 37 • C for 1 h. After that, the tubes were centrifuged at 4500 rpm for 15 min and washed twice in PBS. The cells were resuspended in PBS containing crystal violet (10 µg/mL). After 15 min, the tubes were incubated at 37 • C and centrifuged (15 min, 4500 rpm), and the absorbance of the supernatant at 595 nm was measured. The experiment was repeated five times, and the results were averaged. The percentage of crystal violet uptake was evaluated according to the equation The human THP-1 cell line (European Collection of Cell Cultures, Salisbury, UK) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% Lglutamine, 1% penicillin, and streptomycin at 37 • C with 5% CO 2 . Cells were passaged at approximately 1-week intervals [70]. All the tested compounds were prepared and dissolved as mentioned in our previous work [22]. The preliminary in vitro screening of the cytotoxicity of the most effective compounds was performed using the human THP-1 cell line, as described previously [22]. The cytotoxicity was evaluated after 24 h as the IC 50 value (compound concentration causing 50% inhibition of cell population proliferation), see Table 2.

Conclusions
Pattern 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide and the thirteen carbamates derived from it were prepared and characterized. All the compounds were tested against S. aureus ATCC 29213 and E. faecalis ATCC 29212 as the references and quality control strains. While the compounds did not have anti-enterococcal activity, they showed remarkable antistaphylococcal activity, not only against S. aureus but also against MRSA isolates at the nanomolar and submicromolar levels. Although the primary in vitro cytotoxicity performed on the human cancer cell line THP-1 was in the IC 50 range of 2.61-3.44 µM, the calculated selectivity index for only three compounds was <20. 2-[(2,4,5-Trichlorophenyl) carbamoyl]naphthalen-1-yl ethylcarbamate (2) and 2-[(2,4,5-trichlorophenyl)carbamoyl] naphthalen-1-yl butylcarbamate (4) showed MICs in the range of 18-64 nM against S. aureus and at least two other MRSA isolates, while 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl heptylcarbamate (7) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl (4-phenylbutyl)carbamate (14) demonstrated MBCs in the range of 0.124-0.461 µM against all the tested staphylococci. It can be summarized that the MIC and MBC effects against S. aureus decrease with increasing lipophilicity and bulkiness of the carbamate tails, while against most of the MRSA isolates the bactericidal activity increases with increasing lipophilicity and length of the carbamate tail, up to C (7) , or phenylbutyl chain, and then sharply decreases due to the cut-off effect. The crystal violet uptake assay did not show significant damage to the bacterial wall/membrane. Rather, interactions of the compounds with the bacterial enzyme equipment can be predicted, as demonstrated by the MTT assay. Based on all these observations, it can be concluded that most of the tested carbamates can be considered as promising agents suitable for further study. However, the results obtained in this study will need to be supported by functional genomic and proteomic studies to reveal the mechanism of action of carbamates on staphylococci.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15060715/s1, Figure S1: Mutual comparison of experimentally determined values of log k, log D 6.5 and log D 7.4 of all prepared compounds 1-14; Figure S2: Comparison of experimentally determined values of log k, log D 6.5 and log D 7.4 of prepared compounds with predicted Clog P (ChemDrawUltra) values; Figure S3: Comparison of experimentally determined values of log k, log D 6.5 and log D 7.4 of prepared compounds with predicted log P (ACD/Percepta) values; Figure S4: Comparison of experimentally determined values of log k, log D 6.5 and log D 7.4 of prepared compounds with predicted log P (ChemDrawUltra) values; Figure