A Novel Class of Cationic and Non-Peptidic Small Molecules as Hits for the Development of Antimicrobial Agents

Cationic and non-peptide small molecules containing a total of six positive charges arranged on one side and a long aliphatic tail on the other have been synthesized and tested against Gram-positive and Gram-negative bacteria. The positive charges have been contributed by two aminophenol residues. These molecules have showed remarkable antimicrobial activity against Gram-positive bacteria including multidrug-resistant strains. Our structure–activity relationship studies demonstrated the importance of the length and flexibility of the hydrophobic tail for the antimicrobial activity. Importantly, these compounds are non-toxic to eukaryotic cells at the concentration affecting growth in bacteria, reflecting an acceptable margin of safety. The small size and easy synthetic accessibility of our molecules can be of interest for the further development of novel antimicrobials against Gram-positive bacterial pathogens, including multidrug-resistant strains.


Introduction
At present, microbial resistance to conventional antibiotics is becoming a growing problem that affects, in particular, to hospitals and other health care centers. This crisis has been attributed to the failure to correctly administrate antibiotics or to strictly use them when necessary, to feed farm animals with antibiotics as well as to the lack of investment of the pharmaceutical companies in the development of new antibiotics [1][2][3].
According to a recent report of the World Health Organization, if no initiatives are taken the cost of resistance to antibiotics could exceed $100 billion and lead to the premature death of 300 million people in 2050 [4]. Therefore, it is of critical importance to develop new antimicrobial agents in order to substitute or complement currently available antibiotics.
It is well known that many multicellular organisms (animal and plants) produce a variety of peptides, namely antimicrobial peptides (AMPs) and host defense peptides (HDPs), which are effective defensive weapons against a wide range of pathogens including, bacteria, fungi, enveloped viruses, and protozoa [5,6].
Naturally occurring AMPs are amphipathic molecules having both hydrophobic residues and hydrophilic positive charges (cationic amino acids), which allow them to bind simultaneously at several sites on the biological membranes [5][6][7][8]. In the particular case of bacteria, the main driving force leading to an efficient binding of these amphipathic molecules is the interaction between the positive charges of AMPs and the negatively charged phospholipid head-groups present on the surface of bacterial membranes [9][10][11][12]. This is followed by interaction of the hydrophobic residues of AMPs with the lipid component of the bacterial membrane, leading to its perturbation, and in certain cases, internalization of the peptide damaging critical intracellular targets [5,6,9]. The ability of AMPs to distinguish between bacterial and mammalian cells is mainly due to the differences in the lipid components of their respective cell membranes. In bacteria, the surface exposed to the outer world is heavily populated by lipids with negatively charged phospholipid head-groups, while the membranes of plant and animals is composed principally of lipids with no net charge [5,[9][10][11][12].
With respect to conventional antibiotics, AMPs showed several advantages. First, the emergence of resistance against AMPs is less probable than in the case of conventional antibiotics [5,12,13]. In addition, AMPs are able to modulate the immune response though a variety of mechanisms to fight infections [5,6,9,[14][15][16]. Moreover, AMPs also show a broad antimicrobial spectrum acting against a variety of pathogens other than bacteria such as viruses, fungi, and protozoa [5,[14][15][16]. Despite all of these advantages, AMPs also show several disadvantages that limit their clinical use. Among them, poor bioavailability, potential lability to proteases and high cost of manufacturing [6]. In fact, although some AMPs are currently in clinical trials, there are still few of them available for clinical use-those being polymyxin B, colistin, gramicidin S, daptomycin, and nisin [17,18].
Inspired by the antimicrobial activities showed by the natural AMPs, different synthetic mimics have been described over the past few years. Most of them are peptidomimetics but also polymers or oligomers (molecular weight > 1000 Da) [19,20]. With the aim of overcoming the problems associated with the large size of these molecules, more manageable downsized compounds (molecular weight < 1000 Da) have been also synthesized [21][22][23][24][25].
Having all of this in mind, we decided to synthesize small molecules (molecular weight < 1000 Da) of general formula I ( Figure 1) as AMPs mimics. These synthetic molecules bear two aminophenol 'heads' on one side and a long aliphatic tail of different length on the other. In the design of these small molecules, some essential structural characteristics of the natural AMPs, such as the simultaneous presence of cationic charges and hydrophobic groups, have been taken into consideration. In our case, the hydrophobicity was provided by the two aromatic rings of the 'heads' and the length of the tail, while hydrophilicity by the six positive charges (+6) of the quaternary ammonium groups of the 'heads'. The length of the alkyl chain, -(CH 2 ) n CH 3 , was varied systematically through variation of the number of methylene groups (n = 6 to n = 16). Finally, in order to determine the role of the conformational flexibility in the antibacterial activity, analogues in which the aliphatic 'tail' has been replaced with an unsaturated chain with one or four double bonds were prepared.
For these compounds, inhibition of representative drug-sensitive and multidrug-resistant bacterial pathogens has been determined and is herein described.

Chemical Results
The synthesis of the proposed final compounds was achieved in four steps. The aromatic 'head' 3, containing three Boc-protected amino groups was prepared first (Scheme 1). With this purpose, commercially available methyl 3,4,5-trihydroxybenzoate 1 was treated with 2-(Boc-amino) ethyl bromide in the presence of NaI and Cs2CO3 (Scheme 1) to afford intermediate 2 (69%). Saponification of the methyl ester present on 2, using LiOH/H2O, afforded compound 3 (84% yield), with a free carboxylic acid on the focal point (Scheme 1). It should be mentioned that the synthesis of 2 has been previously described [26] using potassium carbonate (K2CO3) as a base. In our hands, this method led to a poor yield of this compound (10%). However, when cesium carbonate and sodium iodide were used, the yield of 2 improved until 69%. Next, reaction of the commercially available 2-amino-1,3-propanediol (serinol) 4 (Scheme 2) with the corresponding acyl chloride, in the presence of trimethylamine at −20 °C, afforded the N-acyl intermediates 5-9 in moderate to good yields (57-89%).
Catalytic hydrogenation of 13 at atmospheric pressure, in the presence of 10% Pd/C in ethyl acetate, afforded the saturated derivative 15 in quantitative yield (Scheme 4).

Chemical Results
The synthesis of the proposed final compounds was achieved in four steps. The aromatic 'head' 3, containing three Boc-protected amino groups was prepared first (Scheme 1). With this purpose, commercially available methyl 3,4,5-trihydroxybenzoate 1 was treated with 2-(Boc-amino) ethyl bromide in the presence of NaI and Cs 2 CO 3 (Scheme 1) to afford intermediate 2 (69%). Saponification of the methyl ester present on 2, using LiOH/H 2 O, afforded compound 3 (84% yield), with a free carboxylic acid on the focal point (Scheme 1). It should be mentioned that the synthesis of 2 has been previously described [26] using potassium carbonate (K 2 CO 3 ) as a base. In our hands, this method led to a poor yield of this compound (10%). However, when cesium carbonate and sodium iodide were used, the yield of 2 improved until 69%.

Chemical Results
The synthesis of the proposed final compounds was achieved in four steps. The aromatic 'head' 3, containing three Boc-protected amino groups was prepared first (Scheme 1). With this purpose, commercially available methyl 3,4,5-trihydroxybenzoate 1 was treated with 2-(Boc-amino) ethyl bromide in the presence of NaI and Cs2CO3 (Scheme 1) to afford intermediate 2 (69%). Saponification of the methyl ester present on 2, using LiOH/H2O, afforded compound 3 (84% yield), with a free carboxylic acid on the focal point (Scheme 1). It should be mentioned that the synthesis of 2 has been previously described [26] using potassium carbonate (K2CO3) as a base. In our hands, this method led to a poor yield of this compound (10%). However, when cesium carbonate and sodium iodide were used, the yield of 2 improved until 69%. Next, reaction of the commercially available 2-amino-1,3-propanediol (serinol) 4 (Scheme 2) with the corresponding acyl chloride, in the presence of trimethylamine at −20 °C, afforded the N-acyl intermediates 5-9 in moderate to good yields (57-89%).
Catalytic hydrogenation of 13 at atmospheric pressure, in the presence of 10% Pd/C in ethyl acetate, afforded the saturated derivative 15 in quantitative yield (Scheme 4).
Initial assays performed in liquid culture showed that none of these compounds 16-21 proved inhibitory against the Gram-negative bacteria S. Typhimurium SV5015 up to 50 µg mL −1 (Figure 2A). However, with the exception of 16, the rest of compounds 17-21 showed inhibitory effects against the Gram-positive bacteria L. monocytogenes EGD-e (only 20, as representative of the active compounds, is showed in Figure 2B). Interestingly, 17-21 also showed inhibitory effects against the multidrug-resistant bacteria S. aureus USA-300 (only 20, as representative of the active compounds, is showed in Figure 2C).
Initial assays performed in liquid culture showed that none of these compounds 16-21 proved inhibitory against the Gram-negative bacteria S. Typhimurium SV5015 up to 50 µg mL −1 (Figure 2A). However, with the exception of 16, the rest of compounds 17-21 showed inhibitory effects against the Gram-positive bacteria L. monocytogenes EGD-e (only 20, as representative of the active compounds, is showed in Figure 2B). Interestingly, 17-21 also showed inhibitory effects against the multidrug-resistant bacteria S. aureus USA-300 (only 20, as representative of the active compounds, is showed in Figure 2C).  Next, the minimal inhibitory concentrations (MIC) of 16-21 have been determined. In this experiment, kanamycin-a conventional aminoglycoside antibiotic that inhibits the synthesis of bacterial proteins-was used as control [27]. Table 1 summarizes the results of this evaluation. As it was shown in Table 1, the N-octanoyl derivative 16, with an acyl chain containing only 6 methylenes, did not show significant activity up to 50 µg mL −1 against any of the drug-sensitive bacteria (Salmonella enterica serovar Typhimurium strain SV5015, Listeria monocytogenes strain EGD-e, and Staphylococcus aureus strain Newman). The N-dodecanoyl analogue, compound 17, with 10 methylenes, displayed improved antibacterial activity against L. monocytogenes EGD-e and S. aureus Newman (MIC: 12.5 and 50 µg mL −1 , respectively). Further increase in the length of the aliphatic chain (14 methylenes) yielded compound 18 (N-hexadecanoyl analogue) that displayed much improved antibacterial activity against L. monocytogenes EGD-e and S. aureus Newman (MIC: 3.13 and 12.5 µg mL −1 , respectively). However, the highest long chain analogue 19 (N-octadecanoyl analogue, 16 methylenes) did not display any significant change in activity with respect to 18, indicating that the additional two methylene groups present in the aliphatic chain of 19 are superfluous.
The effect of increasing the rigidity of the molecule in the antibacterial activity was analyzed with compounds 20 and 21, with one and four double bonds, respectively, in the aliphatic chain. The unsaturated analogue 20, with only one double bond in the aliphatic chain, was found to be as active against L. monocytogenes EGD-e and S. aureus Newman as the saturated counterparts 18 and 19, whereas compound 21, containing four double bonds was less active, suggesting that an excessive conformational rigidity in the aliphatic chain is detrimental for the antibacterial activity.
The new aminophenol derivatives 16-21 were also tested for their in vitro inhibitory effects on clinically isolated multidrug-resistant (MDR) bacteria such as S. aureus strains SC-1 and USA-300 [28].
Compounds 17 and 21 only showed moderate activity (MIC: 50 µg mL −1 ) against the drug-resistant strain S. aureus SC-1, while they showed significant activity (MIC: 12.5 µg mL −1 ) against the drug-resistant strain S. aureus USA-300. Just the opposite was observed for compound 19, which displayed moderate activity (MIC: 50 µg mL −1 ) against S. aureus USA-300 strain and significant (MIC: 12.5 µg mL −1 ) against S. aureus SC-1. Interestingly, the activity showed by compounds 18 and 20 against both resistant strains was remarkable. It contrasted with the lack of activity showed by the conventional antibiotic kanamycin, which was used as control. Similar to the results found for the drug-sensitive strains, compound 16, with the shortest aliphatic chain (octanoyl chain), did not show significant activity up to 50 µg mL −1 against any of the multidrug-resistant bacteria.
The saturated compound 18, with a long aliphatic chain (C16) and 20 with a long chain (C18) containing only one unsaturation, were therefore the most potent compounds of this series against all the tested bacteria strains (drug-sensitive and multidrug-resistant).
It should be noted that the activity showed by 18 and 20 against Gram-positive drug-sensitive bacteria is very similar to that of the conventional antibiotic used as control, kanamycin. Moreover, 18 and 20 resulted active against drug-resistant bacterial strains for which kanamycin was inactive.
In summary, the structure-activity relationship (SAR) studies reveal the significance of our design for achieving Gram-positive antimicrobial activity, especially against drug-resistant bacterial strains. Our molecules, that display the most important characteristic of most AMPs, like cationic facial amphiphilicity and positive charges, have several advantages over the natural counterparts. Among them, non-peptidic character, low-cost synthesis, and possibility to fine-tuning their potency and safety. Based on that, they can be useful hits for the further design of novel antimicrobial agents against Gram-positive bacteria.

Antimicrobial Kinetics
Next, the effect of the newly synthesized compounds over the bacterial growth in function of time was determined. Bacterial growth (cells per volume unit), estimated from the turbidity of the culture, was measured using a spectrophotometer at a wavelength of 600 nm and it was represented as optical density (OD 600 ).
First, we monitored the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e in the presence of compounds 17, 19, and 20, used at a concentration that was four-fold higher than their respective MICs (4 MIC) (see Table 1). The aminoglycoside kanamycin at 30 µg mL −1 was used as relevant control since it rapidly arrests growth in actively proliferating bacteria [27]. As it was shown in Figure 3A the curves for compounds 17, 19, and 20 are very similar to those of the kanamycin. Thus, this experiment clearly shows that the treatment with aminophenols 17, 19, and 20 resulted in rapid arrest of bacteria growth ( Figure 3A) and, consequently, these compounds behave as potent antimicrobials. The saturated compound 18, with a long aliphatic chain (C16) and 20 with a long chain (C18) containing only one unsaturation, were therefore the most potent compounds of this series against all the tested bacteria strains (drug-sensitive and multidrug-resistant).
It should be noted that the activity showed by 18 and 20 against Gram-positive drug-sensitive bacteria is very similar to that of the conventional antibiotic used as control, kanamycin. Moreover, 18 and 20 resulted active against drug-resistant bacterial strains for which kanamycin was inactive.
In summary, the structure-activity relationship (SAR) studies reveal the significance of our design for achieving Gram-positive antimicrobial activity, especially against drug-resistant bacterial strains. Our molecules, that display the most important characteristic of most AMPs, like cationic facial amphiphilicity and positive charges, have several advantages over the natural counterparts. Among them, non-peptidic character, low-cost synthesis, and possibility to fine-tuning their potency and safety. Based on that, they can be useful hits for the further design of novel antimicrobial agents against Gram-positive bacteria.

Antimicrobial Kinetics
Next, the effect of the newly synthesized compounds over the bacterial growth in function of time was determined. Bacterial growth (cells per volume unit), estimated from the turbidity of the culture, was measured using a spectrophotometer at a wavelength of 600 nm and it was represented as optical density (OD600).
First, we monitored the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e in the presence of compounds 17, 19, and 20, used at a concentration that was four-fold higher than their respective MICs (4 MIC) (see Table 1). The aminoglycoside kanamycin at 30 µg mL −1 was used as relevant control since it rapidly arrests growth in actively proliferating bacteria [27]. As it was shown in Figure 3A the curves for compounds 17, 19, and 20 are very similar to those of the kanamycin. Thus, this experiment clearly shows that the treatment with aminophenols 17, 19, and 20 resulted in rapid arrest of bacteria growth ( Figure 3A) and, consequently, these compounds behave as potent antimicrobials.  Table 1); (B) Growth curves of L. monocytogenes strain EGD-e and S. aureus cultures exposed to the amino phenol 18. Amino phenol 18 was used at four-fold of its respective MIC value for each of the indicated bacterial strains (see Table 1).  Table 1); (B) Growth curves of L. monocytogenes strain EGD-e and S. aureus cultures exposed to the amino phenol 18. Amino phenol 18 was used at four-fold of its respective MIC value for each of the indicated bacterial strains (see Table 1). Next, the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e and S. aureus Newman, together with the multidrug-resistant bacteria S. aureus strains SC1 and USA300 was analyzed in the presence of the most potent aminophenol derivative 18 (4 MIC) ( Figure 3B). Our results showed that 18 is able of arresting the growth of the four studied bacterial strains. Apparently, S. aureus Newman (drug-sensitive) and S. aureus SC-1 (multidrug-resistant) strains were more sensitive to the action of 18 than the other bacterial strains ( Figure 3B).

Cytotoxicity Evaluation
Finally, the cytotoxic effects of the aminophenol derivatives 17-21 were determined in two different eukaryotic cell types, HeLa epithelial cells ( Figure 4) and NRK-49F fibroblasts ( Figure 5). Next, the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e and S. aureus Newman, together with the multidrug-resistant bacteria S. aureus strains SC1 and USA300 was analyzed in the presence of the most potent aminophenol derivative 18 (4 MIC) ( Figure 3B). Our results showed that 18 is able of arresting the growth of the four studied bacterial strains. Apparently, S. aureus Newman (drug-sensitive) and S. aureus SC-1 (multidrug-resistant) strains were more sensitive to the action of 18 than the other bacterial strains ( Figure 3B).

Cytotoxicity Evaluation
Finally, the cytotoxic effects of the aminophenol derivatives 17-21 were determined in two different eukaryotic cell types, HeLa epithelial cells ( Figure 4) and NRK-49F fibroblasts ( Figure 5). After an overnight exposure to the compounds, toxicity-denoted by detachment of cells and drastic morphological changes-was evident only at 100 µg mL −1 while no toxicity was found at lower concentrations. Importantly, the highest concentration (100 µg mL −1 ) was 8-fold or 32-fold higher than the MIC value observed for 17-21 in the antimicrobial assays (see Table 1) reflecting an acceptable margin of safety. After an overnight exposure to the compounds, toxicity-denoted by detachment of cells and drastic morphological changes-was evident only at 100 µg mL −1 while no toxicity was found at lower concentrations. Importantly, the highest concentration (100 µg mL −1 ) was 8-fold or 32-fold higher than the MIC value observed for 17-21 in the antimicrobial assays (see Table 1) reflecting an acceptable margin of safety.

Conclusions
In this study, we have developed an efficient strategy for the synthesis of novel aminophenol derivatives 17-21. All of these compounds have the same hydrophilic portion consisting in two cationic 'heads' with three positive charges each (six positive charges in total) and a lipophilic 'tail' of different length and conformational flexibility. These compounds were synthesized in four simple synthetic steps.
The synthesized compounds showed antimicrobial activity against different Gram-positive bacteria (L. monocytogenes and S. aureus) while probed to be inactive against Gram negative bacteria (S. Typhimurium). Interestingly 17-21 also showed inhibitory effects against Gram-positive multidrug-resistant strains such as S. aureus SC-1 and S. aureus USA-300.
In this respect, it should be noted that defined AMPs have been reported to be only active against Gram-positive bacteria while others are not selective and showed broad activity against both Gramnegative and Gram-positive bacteria [29][30][31]. Despite their mode of action not being fully understood and the basis for their selectivity being unknown, many studies reveal the importance of the membrane components of bacteria-lipid content in particular-in the activity and selectivity of these molecules [29][30][31].
These precedents support the idea of a mechanism of action for the compounds here described directly related to a defined structure in the bacterial cell envelope. Further studies are required to decipher their mode of action and selectivity towards microbial cells.

Conclusions
In this study, we have developed an efficient strategy for the synthesis of novel aminophenol derivatives 17-21. All of these compounds have the same hydrophilic portion consisting in two cationic 'heads' with three positive charges each (six positive charges in total) and a lipophilic 'tail' of different length and conformational flexibility. These compounds were synthesized in four simple synthetic steps.
The synthesized compounds showed antimicrobial activity against different Gram-positive bacteria (L. monocytogenes and S. aureus) while probed to be inactive against Gram negative bacteria (S. Typhimurium). Interestingly 17-21 also showed inhibitory effects against Gram-positive multidrug-resistant strains such as S. aureus SC-1 and S. aureus USA-300.
In this respect, it should be noted that defined AMPs have been reported to be only active against Gram-positive bacteria while others are not selective and showed broad activity against both Gram-negative and Gram-positive bacteria [29][30][31]. Despite their mode of action not being fully understood and the basis for their selectivity being unknown, many studies reveal the importance of the membrane components of bacteria-lipid content in particular-in the activity and selectivity of these molecules [29][30][31].
These precedents support the idea of a mechanism of action for the compounds here described directly related to a defined structure in the bacterial cell envelope. Further studies are required to decipher their mode of action and selectivity towards microbial cells.
Presence of long chains (C16 or C18) is an important structural requirement for antimicrobial activity. Introduction of one double bond in a long alkyl chain (C18) renders molecules that also showed good antibacterial activity while the incorporation of four double bonds led to much less active molecules. This finding emphasized the importance of the length and flexibility of the hydrophobic alkyl portion ('tail') for the antibacterial activity.
Compounds 18 (C16) and 19 (C18) with saturated long chains, together with 20, that incorporates one double bond in its long chain (C18), were found to be the most potent antibacterial agents among this series. These compounds are non-toxic to eukaryotic cells at the concentration affecting growth in bacteria, reflecting an acceptable margin of safety.
Taken together, these data proved that the newly synthesized aminophenol compounds can be promising hits for the further development of antimicrobial agents against Gram-positive bacteria (drug-sensitive and multidrug-resistant). However, further research is needed to elucidate the pharmacophore group(s) and their mechanism of action.

Synthesis
Commercial reagents and solvents were used as received from the suppliers without further purification unless otherwise stated. The solvents used in some reactions were dried prior use. DMF dry was commercially available (Sigma-Aldrich Quimica SL, Madrid, Spain).
Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-coated with silica gel 60 (F 254 , 0.25 mm). Products were visualized using an ultraviolet lamp (254 and 365 nm) or by heating on a hot plate (approx. 200 • C), directly or after treatment with a 5% solution of phosphomolybdic acid or vanillin in ethanol.
For HPLC analysis, an Agilent Technologies 1120 Compact LC with a reverse phase column ACE 5 C18-300 (4.6 × 150 mm, 3.5 µm) equipped with a PDA (photodiode array) detector Waters 2996 was used. Acetonitrile was used as mobile phase A, and water with 0.05% of TFA was used as mobile phase B at a flow rate of 1 mL min −1 . All retention times are quoted in minutes and the gradients are specified for each compound in the experimental data.
According to the general procedure, serinol 4 (49 mg, 0.54 mmol) was treated with the above mentioned arachidonic chloride (193 mg, 0.6 mmol) and TEA (0.1 mL) to give 182 mg (89%) of 9 as an amorphous white solid. 1  A solution of the Boc protected gallic acid derivative 3 (2.2 eq), DMAP (1.6 eq), and DCC (3.2 eq) in dry CH 2 Cl 2 (7.5 mL) was stirred at room temperature for 15 min and then added dropwise to a second solution, also stirred for 15 min, containing the corresponding serinol derivate 5-9 (1 eq) and DMAP (1.6 eq) in dry CH 2 Cl 2 (7.5 mL). To dissolve all components, an extra amount of DMF (2 mL) were added. The solution was stirred at 30 • C for 24 h and then evaporated to dryness. The residue was dissolved in ethyl acetate (20 mL). Urea by-product was filtered and the organic residue was evaporated. This process was repeated several times until no precipitation was observed. The residue was purified on a Biotage HPFC purification system in a reverse phase using water:acetonitrile (100:0 to 0:100) to give the corresponding intermediate. Note that all the solid reactives are pre-dried overnight by storage inside a vacuum desiccator with a drying agent like phosphorous pentoxide.  (11) Following the general procedure, 6 (50 mg, 0.2 mmol) was treated with 3 (240 mg, 0.4 mmol) to afford 112.5 mg (43%) of 11 as an amorphous white solid. 1 (12) Following the general procedure, 7 (25 mg, 0.07 mmol) was treated with 3 (127 mg, 0.21 mmol) to afford 11.8 mg (10%) of 12 as an amorphous white solid. 1  To a solution containing the corresponding Boc protected derivative (10)(11)(12)(13)(14)(15) in CH 2 Cl 2 (10 mL), TFA was added. After stirring at room temperature overnight the solution was evaporated to dryness and co-evaporated successively with CH 2 Cl 2 and MeOH. The residue was lyophilized to afford the final products.

Monitoring of Bacterial Growth in the Presence of Aminophenol Compounds
Overnight cultures were diluted 1:100 (L. monocytogenes) or 1:500 (S. aureus) in fresh medium. After aprox. 1.5-2 h of incubation when cultures reached an OD 600~0 .2, bacteria were exposed to four-fold the MIC value of the specific antimicrobial to be tested. OD 600 where then measured every 30 min.

Toxicity Assays in Eukaryotic Cell Cultures
Cultures of normal rat fibroblasts NRK-49F (ATCC CRL-1570) and HeLa human epithelial cells (ATCC CCL-2) were used to assess putative toxicity effects of the aminophenol-derivate compounds. The cells were seeded in 24-well plates and propagated in Dulbecco's modified Eagle's medium (DMEM) or Eagle's minimum essential medium containing 10% (v/v) fetal bovine serum (FBS) at 37 • C in a 5% CO 2 atmosphere. Following adherence of the cells to the substrate, the medium was replaced with fresh medium containing different concentrations of the compounds. To 0.5 mL of tissue culture medium per well either 5 µL to 1.25 µL of a 10 mg mL −1 stock solution of the compound to test, were added. This was equivalent to a final concentration of 100 to 20 µg mL −1 , respectively. A control containing 5 µL of the solvent DMSO was analyzed in parallel. The toxicity potential of the compounds was evaluated based on two main parameters: morphological changes in the fibroblast or epithelial cells and fraction of the monolayer culture that lost adherence to the substrate. Changes in these parameters were monitored by microscopy. Cells were fixed with 3% (v/v) paraformaldehyde (PFA) and processed as described [37]. Images were acquired on an inverted Leica DMI 6000B microscope with an automated CTR/7000 HS controller (Leica Microsystems, Wetzlar, Germany) and an Orca-R2 CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).

Phase-Contrast Microscopy
To determine probable effect of the aminophenol-derivate compounds in bacterial morphology, bacteria were grown to mid-exponential phase (OD600~0.2-0.3). At this time, bacteria were exposed for 1 h to a concentration of the compound equivalent to 4 MIC and the OD 600 measured every 30 min. To monitor probable morphological changes not appreciable by OD 600 , the bacteria were harvested by centrifugation (4300× g, 5 min, RT), washed in PBS, fixed with 3% (v/v) PFA, and processed for microscopy as described [38].
Supplementary Materials: Supplementary materials are available online. Copies of representative 1 H and 13 C NMR spectra are included.