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Aminobenzylated 4-Nitrophenols as Antibacterial Agents Obtained from 5-Nitrosalicylaldehyde through a Petasis Borono–Mannich Reaction

Multidrug-resistant bacteria are one of the current biggest threats to public health and are responsible for most nosocomial infections. Herein, we report the efficient and facile synthesis of antibacterial agents aminoalkylphenols, derived from 5-nitrosalicyladehyde and prepared through a Petasis borono–Mannich multicomponent reaction. Minimum inhibitory concentrations (MICs) as low as 1.23 μM for a chlorine derivative were determined for multidrug-resistant Gram-positive bacteria, namely, Staphylococcus aureus and Enterococcus faecalis, two of the main pathogens responsible for infections in a hospital environment. The most promising antibacterial agents were further tested against eight strains of four Gram-positive species in order to elucidate their antibacterial broadness. In vitro cytotoxicity assays of the most active aminoalkylphenol revealed considerably lower toxicity against mammalian cells, as concentrations one order of magnitude higher than the determined MICs were required to induce human keratinocyte cell death. The phenol moiety was verified to be important in deeming the antibacterial properties of the analyzed compounds, although no correlation between such properties and their antioxidant activity was observed. A density functional theory computational study substantiated the ability of aminoalkylphenols to serve as precursors of ortho-quinone methides.


■ INTRODUCTION
The discovery and development of antibiotics stands as one of mankind's greatest achievements. However, the number of infections provoked by multidrug-resistant bacteria is increasing at a remarkable pace, a problem that science has not been able to address. 1 The unrestrained use of antibiotics in the last 50 years has been advocated as one of the reasons for the colonization and infection due to drug resistant bacteria. 2,3 It is estimated that in Europe and the United States, 48 000 people die each year because of multidrug-resistant bacterial infections, 4 and that in 2016, there were 600 000 worldwide cases with resistance to rifampicin, of which 490 000 had multidrug-resistant tuberculosis. 5 If no actions are taken to tackle this severe public health issue, it is foreseen that by 2050, the death toll can rise up to 10 million lives when considering drug resistance of only six pathogens. 6 This is even more alarming in the hospital environment or other health care facilities, where acquired infections are associated with significant morbidity and mortality, additional health care expenditure, greater use of broad spectrum antibiotics (which amplifies the emergence and reemergence of drug resistant microorganisms), and increased costs. 7 According to WHO estimates, of every hundred hospitalized patients, 7 in developed and 10 in developing countries will acquire at least one nosocomial infection. 8 Besides intrinsic patient factors (age, duration of hospitalization, or underlying diseases), many extrinsic factors, such as caregivers' practices, surgical operations, the use of invasive devices, administration of broad-spectrum antibiotics, or immunosuppressive agents, are risk predisposers to these infections. 9, 10 The majority of nosocomial infections are caused by bacteria, with Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus, and Streptococcus spp. leading. 11 Despite the society being in urgent need for new antibacterial agents, the small fraction of yearly revenue per patient generated by such agents when compared with anticancer drugs have pushed the pharmaceutical companies away from their research and development programs on antibiotics. 12,13 Opportunely, the development of antibiotics has been relaunched by small biotech companies, and alternatives to antibiotics, including "non-compound" approaches and small molecule "resistance breakers", are growing trends in the field. 14 Motivated by the antimicrobial activity of salicylaldehydes 15−17 and their Schiff bases, 18 we have recently reported the preparation and antimicrobial screening of several aminoalkylphenols. 19 From screening of a library of 43 compounds, some structural features pivotal for the antibacterial activity have been identified, namely, indoline as the amine counterpart and a para-nitrophenol group. Promising antibacterial activity against several resistant microorganisms such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE), as well as other nonpathogenic Gram-positive strains, was reported for some of such derivatives. Notwithstanding the fact that aminoalkylphenols can be problematic compounds in highthroughput screenings because of their ability to form reactive quinone methides or acting as metal chelators, 20,21 their anticancer and cytotoxic properties continue to be extensively explored. 22,23 Almost simultaneously to our report, Roman and co-workers reported the antibacterial properties of 1-aminoalkyl 2-naphthols, in which similar properties were observed against Gram-positive bacteria. 24  requirements for antibacterial activity were determined by Roman, as thiophen-2-yl derivatives showed the best activity, regardless of the nature of the amine moiety. The antibacterial properties of other Mannich bases derived from naphthol have been previously explored. 25 Aminoalkylphenols have also been reported to inhibit biofilm formation 26 and classified as antiinfectives rather than traditional antibiotics as they cure Caenorhabditis elegans of an Enterococcus faecalis infection at significantly lower concentration than the one required for in vitro bacteria growth inhibition. 27 After observing that the concentration required for an antibacterial effect of some promising aminoalkylphenols was generally inferior to the cytotoxic concentrations, 19 we then set to expand our previous library in order to optimize the antibacterial properties.

■ RESULTS AND DISCUSSION
As multicomponent reactions are a remarkable tool for the easy preparation of libraries of compounds, 28,29 our previous library of aminoalkylphenols was expanded using the Petasis borono−Mannich 30,31 reaction (Scheme 1). Starting from 5nitrosalicylaldehyde 1 and indoline 2, the corresponding iminium was formed in situ and trapped with different aryl boronic acids 3 to provide the desired tertiary amines 4−17.
Ether 18 was prepared by methylation of the phenol functionality upon treatment of 4 with dimethyl sulfate. Tertiary amine 21, lacking the oxygen functionality in the nitroaryl substituent, was prepared by Friedel−Crafts acylation of toluene with meta-nitro benzoyl chloride 19 followed by reductive amination with indoline. The selection of compounds to be prepared was reasoned based on two aspects, first, by replacing the 4-methyl substituent in 4 with other isosteres because we have previously identified position 4 of the phenyl moiety to be important in conferring the desired antibacterial activity. Therefore, the 4-methyl substituent in 4 was replaced by other alkyl substituents or heteroatoms such as thioether, hydroxyl, carbamate, and halogens. Second, in order to fully disclose the importance of the phenol moiety in the nitroaryl moiety, the methyl ether derivative 18 and tertiary amine 21 were also prepared and their antibacterial properties were evaluated.
The synthesized aminoalkylphenols 4−17 and derivatives 18 and 21 were tested against a large panel of Gram-positive microorganisms (Table 1), namely, three S. aureus strains (a reference ATCC, a methicillin-resistant and a non-resistant strain) and two E. faecalis strains (a reference ATCC and a vancomycin-resistant strain). An E. coli strain, representative of Gram-negative microorganisms, was also tested, although no antibacterial properties were observed for any of the compounds assayed (not shown). The modification of the para-methyl substituent to other alkyl substituents resulted in derivatives 6−8 of a similar antibacterial profile, except when a longer C 6 alkyl chain was introduced in 9. Replacing the alkyl substituent by a heteroatom functionality such as thioether, alcohol, or a carbamate also had a detrimental effect, as observed for 10−12, respectively. Gladly, the introduction of a halide such as chloride (13) or bromide (14) resulted in not only augmenting the bacterial inhibition growth but also in efficacy of killing the bacteria. Notably, a more effective activity was determined for chloro derivative 13, for which minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) as low as 1.23 μM were determined for most of the bacteria tested. On the other hand, the presence of fluoride (15), change of the position of the methyl substituent to the 2-position (16), or replacement of the ptolyl moiety by a 1-naphthyl (17) did not confer any improvement to the antibacterial characteristics of the aminoalkylphenols. Compounds 18 and 21 were poor antibacterials, as the MIC and MBC values increased by one to two orders of magnitude when compared with parental 4, hence evidencing the importance of the phenol functionality in conferring the desired properties.
In order to further elucidate the antibacterial broadness of the most promising synthesized aminoalkylphenols, compounds 4, 5, and 13 were tested against a larger panel of pathogenic Gram-positive bacterial strains with dissimilar resistance phenotypes. Overall, 32 strains from S. aureus, E. faecalis, Streptococcus pneumoniae (S. pneumoniae), and Streptococcus agalactiaegroup B (GBS) (eight clinical isolates from each species) were tested ( Figure 1 and Table S1).
In general, similar MIC values were observed for S. aureus and E. faecalis clinical isolates when compared with those seen for the ATCC adapted strains. Some exceptions occurred, where, at some instances, a maximum of fourfold differences were observed, which may be due both to the dissimilar genetic background of the clinical isolates and to the extensive in vitro passaging of the ATCC strains. As clearly shown in Figure 1, all three selected compounds seem to be much more effective against S. aureus and E. faecalis, displaying a mean of MIC values at least 5.4-fold lower than that observed for S. pneumoniae and S. agalactiae. For the former species, for which the results were encouraging, compound 13 revealed the higher antimicrobial efficacy, given by lower and more homogeneous MIC values.
S. aureus and E. faecalis are among the most important bacteria causing infections in the hospital environment. 32−34 While S. aureus is the primary cause of lower respiratory tract infections and surgical site infections and the second leading cause of nosocomial bacteremia, pneumonia, and cardiovascular infections, E. faecalis is responsible for urinary tract infections (associated with instrumentation and antimicrobial administration) followed by intra-abdominal and pelvic infection, surgical wound infection, bacteremia, endocarditis, neonatal sepsis, and rarely meningitis. 11 Furthermore, because of the large usage of broad-spectrum antibiotics in the hospital environment, both bacteria (mostly S. aureus) are able to emerge and reemerge as multidrug-resistant clones, which complicates the treatment of the caused nosocomial infections. 35,36 A test of lethality to Artemia salina brine shrimp was performed 37 in order to evaluate the toxicity of the different compounds, for which concentrations of 0.1 mg/mL (217− 277 μM) of each compound in dimethylsulfoxide (DMSO) were tested ( Figure 2). Delightfully, a residual 15% mortality rate was observed for compound 13, while compounds 11 and 12 were highly toxic with mortality rates of 89 and 98%, respectively. Fortunately, these compounds were not active against any microorganism on the antibacterial assay. Of the remaining compounds, none held a mortality rate higher than 42%, which was observed for the bromide derivative 14, followed by compound 5 with a 31% mortality rate. For compounds 4 and 5, mortality rates of 21 and 31% were observed, respectively.
To further characterize the toxicity of the compounds, an in vitro model representative of noncancer human cells was also used. The cytotoxicity profile of compound 13 is shown in Figure 3, while other analogues were previously tested 19 using the same protocol. No cytotoxic effects were observed for concentrations of compound 13 up to 26.3 μM. Only the highest concentration tested (65.6 μM) showed relevant cytotoxicity, decreasing cell viability to 53.4 ± 1.5%. Compared with analogue 4 previously tested, for which a concentration of 28 μM led to a cell viability of less than 20%, 19 compound 13 shows a better safety profile.
Taking together, the common feature of the presence of the phenol moiety and the lack of significant antibacterial activity of 18 and 21, it was hypothesized that the high activity observed could be related with the antioxidant properties of phenol compounds. 38 Tests of antioxidant activity of compounds 4−17 using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method ( Figure 4) showed that the bromine and fluorine derivatives 14 and 15 had a scavenging activity of 99 and 94%, respectively. Compounds 4 and 5 are also highly antioxidant (85 and 92%, respectively), although not being the case for the chlorine derivative 13. Furthermore, despite the decent antibacterial properties found for derivatives 6−8, their antioxidant activity was rather low (35−47%), opposing the trend observed for compounds 4 and 5. Interestingly, compound 21, lacking the hydroxyl group, showed antioxidant activity in the same range as the most antioxidant species tested. Therefore, a different main mode of action other than an antioxidant mechanism is likely to be the cause of the antibacterial properties observed.
As the identified aminoalkylphenols bear a stereogenic center in the benzylic position, the separation of both enantiomers of 4 was attempted. Hence, 4 was transformed into the corresponding Mosher's ester 22, separated through preparative thin layer chromatography and further hydrolyzed (Scheme 2). Chiral HPLC analysis of the enantiomerically enriched samples of 4 invariably revealed the presence of significant amounts of a minor enantiomer, which equilibrated to a racemic mixture upon standing in solution. Ultimately, the enantiomerically enriched samples 4a and 4b obtained (with enantiomeric ratios of 65:35 and 16:84, respectively) were also tested for their antibacterial activity (Table 2). Triggered by the difficulty in obtaining enantiomerically pure samples of 4, the putative formation of an ortho-quinone methide (QM) 39,40 was considered. With this in mind, the benzyl alcohol 23, was also prepared by arylation of the 5-nitrosalicylaldehyde (Scheme 2 bottom) and its action for bacteria inhibition growth also tested ( Table 2).  The rather low antimicrobial activity observed for 23 ( Table  2) clearly indicates that the antibacterial properties of 4 are not caused by the hydrolyzed QM. On the other hand, both enantiomerically enriched samples 4a and 4b showed similar levels of toxicity against bacteria as the previously tested racemate. Rokita and co-workers have showed that depending on the electronic feature of the QM, they can be stable toward water nucleophilic attack, while being trapped by nucleotides or other biomolecules. 41,42 Specifically, the introduction of an electron-withdrawing group in the para-phenol position was shown to decrease the rate of the QM formation, while also decreasing its lifetime due to increased reactivity toward hydrolysis. 42 When monitoring by 1 H NMR a solution of 4 in CDCl 3 in the presence of morpholine, we observed the formation of the morpholine derivative in 79% yield after 96 h which was attributed to the chloroform slight acidity ( Figure  S5 in Supporting Information). Contrastingly, a similar experiment in DMSO-d 6 did not show formation of the corresponding morpholine-substituted aminoalkylphenol after 12 h ( Figure S4 in Supporting Information). These observations suggest that even though the nitro substituent can decrease the ability of aminoalkylphenols to serve as QM precursors, their formation and fate is highly dependent on the acidity of the medium. An additional stability test showed that 4 remains stable in DMSO-d 6 at 35°C for at least 11 days ( Figure S6 in Supporting Information). The ability of water and acids in catalyzing nucleophilic attacks to QMs has been demonstrated both theoretically and experimentally. 43−45 Moreover, the presence of an additional aryl substituent should facilitate the formation of the QM because of additional conjugation.
With the previous observations in mind, the formation of two QMs from 24 and 25, the latter lacking the additional phenyl ring and the nitro moiety, was considered by density functional theory (DFT) 46a studies in the gas phase ( Figure  5), by taking into account the previously described role of bulk water in the alkylation of nitrogen nucleophiles. 43,47,48 As expected, the formation of the QMs is a thermodynamically nonfavored process, as the overall ΔG form of QM from 24 is +16.4 and ΔG form of QM from 25 is 25.8 kcal/mol (Figures S1 and S2 in Supporting Information). Comparison of energy profiles in Figures S1−S3 reveals the stabilization effect of the phenyl substituent on the transition states when considering water as a proton shuttle. Additionally, this substituent and the nitro moiety induce the stabilization of the QMs, as the 25derived QM is 10.2 kcal/mol less stable than the one derived from 24. It is worth noting that the presence of the nitro substituent induces a two-step mechanism, as zwitterion species B is a local minimum of the energy profile (see Figure  S3 of Supporting Information for a similar study without the nitro substituent). On the other hand, the formation of the simplest QM from 25 seems to occur by a synchronous process where water acts as a proton shuttle resulting in indoline release. The interaction of the starting tertiary amines   Considering the low activation energy barrier for formation of the 25-derived QM together with their increased stability as compared to the simplest QM, widely used as a nucleotide alkylating agent, 49,50 is reasonable to suggest this path to be responsible for the antibacterial properties of the Mannich bases reported herein. Moreover, several antibiotics, namely, natural products, are known to have their antibacterial properties ground on their capability to form quinone methides and subsequently alkylate macromolecules. 51,52 Contrarily to the recently reported 1-aminoalkyl 2-naphthols by Roman, 24 the antibacterial properties of the compounds described herein are highly dependent on the amine moiety. 19 As indoline is released upon QM formation, other unforeseen modes of action might be the cause of the antibacterial properties. The rather lower cytotoxic levels of the studied compounds are surely dependent on the different metabolisms of the organisms tested and could be explained by a faster kinetic profile toward QM formation in the bacterial media. 53,54 ■ CONCLUSIONS In summary, through use of Petasis borono−Mannich multicomponent reaction, starting from 5-nitrosalicylaldehyde, we were able to expand our library of aminoalkylphenols and find more potent antibacterials than the previously reported. The most active compound synthesized 13 was demonstrated to be selectively active against important bacteria responsible for nosocomial infections (S. aureus and E. faecalis), while only moderately active against S. pneumoniae and S. agalactiae. Gladly, the most active compounds against bacteria showed cytotoxicity against mammalian cells only at concentrations considerably higher than the determined MICs, as verified through an in vitro model. The phenol moiety was verified to be essential to achieve the desired antibacterial properties; however, such properties do not relate with the antioxidant properties of the compounds tested. As Mannich bases have been demonstrated to be precursors of quinone methides, a computational study was performed, and the results found are in line with such a hypothesis. As quinone methides are highly reactive, they can play a role as antibacterial agents, although not encompassing the hydrolyzed adduct. This aspect is also in agreement with the determined MICs of mixtures of different enantiomeric ratios, as both enantiomers can form a similar reactive intermediate. Nevertheless, the antibacterial mode of action of these compounds remains obscure, and further studies will be reported in due course.

■ EXPERIMENTAL SECTION
General Remarks. All reagents were obtained from Sigma-Aldrich or TCI and were used without further purification. The reactions were performed under argon atmosphere and monitored by thin-layer chromatography carried out on precoated (Merck TLC silica gel 60 F254) aluminium plates by using UV light as a visualizing agent and cerium molybdate solution or ninhydrin as developing agents. Flash column chromatography was performed on silica gel 60 (Merck, 0.040−0.063 mm). NMR spectra were recorded with Varian Mercury 300 MHz or JEOL ECZR 500 instruments using CDCl 3 or DMSO-d 6 as solvents and calibrated using tetramethylsilane as internal standard. Chemical shifts (δ) are reported in ppm referenced to the CDCl 3 residual peak (δ 7.26) or TMS peak (δ 0.00) for 1 H NMR, to CDCl 3 (δ 77.16) for 13 C NMR. The following abbreviations were used to describe peak splitting patterns: s = singlet, d = doublet, t = triplet, m = multiplet. Coupling constants, J, were reported in hertz. High-resolution mass spectra were recorded on a Waters ESI-TOF MS spectrometer. Elemental analysis (C, N, and H) was performed on Elementar vario EL III. All compounds tested for antibacterial activity, with exception of 12, were established to be >95% pure upon elemental analysis.
General Method for the Synthesis of Aminoalkylphenols 4−17. Indoline (56 μL, 0.50 mmol) was added to a solution of 2-hydroxy-5-nitrobenzaldehyde (83.6 mg, 0.50 mmol) and arylboronic acid (0.50 mmol) in 1,2-dichloroethane (DCE) or ethanol (EtOH; 5.0 mL) at 50°C. The reaction was stirred at the same temperature until it was complete as judged by TLC. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography to give the pure aminoalkylphenol.
Antibacterial Assays. For selective purposes, the antimicrobial activity of the diverse compounds was first tested against five bacterial Gram-positive strains obtained from American Type Culture Collection (ATCC): S. aureus ATCC25923 (MSSA), S. aureus CIP6538, S. aureus CIP106760 (MRSA), E. faecalis 29212, and E. faecalis ATCC51299 (VRE). For the most promising compounds (i.e., compounds 4, 5, and 13), the antimicrobial activity was further evaluated against eight distinct clinical isolates of S. aureus, E. faecalis, S. pneumoniae, and S. agalactiae (group B) that belong to the wide collection of pathogenic Gram-positive strains of the Portuguese National Institute of Health (NIH).
For each compound, the minimum inhibitory concentration (MIC) was determined by the broth microdilution method, 56 according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. 57 Before each experiment, frozen stocks of all strains were subcultured three times in appropriate culture medium (CAMHB for S. aureus and E. faecalis and CAMHB-LHB 3.5% for streptococci) to check strain viability and to avoid any negative growth effect from congelation, and inocula of 1.5 × 10 8 cfu/mL (the equivalent to 0.5 McFarland) were prepared accordingly. For each microorganism, a 1:20 dilution of the prepared inoculum was used. Twofold serial dilutions of concentrated stock compound solutions (1 mM) were prepared in the required medium into 96-well plates. A control without compounds was also prepared. All cultures were incubated for 16−24 h at 35−37°C with 5% CO 2 . Purity check and colony or viable cell counts of the inoculum suspensions were also evaluated in order to ensure that the final inoculum density closely approximates the intended number. This was obtained by subculturing a diluted aliquot from the growth control well (without compound) immediately after inoculation onto a suitable nonselective agar plate for simultaneous incubation. The MIC was determined as the lowest compound concentration at which no visible growth was observed. The bacterial growth was measured with an absorbance microplate reader (Thermo Scientific Multiskan FC, Loughborough, UK) set to 620 nm.
The MBC was also evaluated. Briefly, after MIC assessment, the bacterial suspension on the wells was homogenized, serialdiluted, triplicate spread on appropriate medium and incubated. The MBC attributed to the compound concentration resulting in a 99.9% reduction in bacterial numbers. All assays were carried out in triplicate for each tested microorganism.
DPPH Method for Antioxidant Activity. The antioxidant activity of the compounds was tested as previously described. 58 To evaluate the compounds' antioxidant potential through the free radical scavenging test, the change in optical density of DPPH radicals was monitored. The absorbance was measured at 517 nm against a corresponding blank and the antioxidant activity was calculated using the following equation The reference standard used for this procedure was butylated hydroxytoluene (BHT).
A. salina Mortality Bioassay. A test of mortality was performed on A. salina brine shrimp, of the compounds 4−17, as previously described. 59 For this assay, an aquarium air pump (HI-FLOTM Single Type 4000), a thermostat Cabinet Aqua Lytic, and a stereomicroscope (CETI Belgium) were used. The number of dead nauplii was recorded after 24 h and used to calculate the percentage (%) of mortality rate, according to the equation Cell Culture and Cytotoxicity Assessment. The cytotoxicity profile of the compound 13 was characterized in the normal-like human keratinocytes (HaCaT cell line). HaCat cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ mL penicillin, and 0.1 mg/mL streptomycin. Cells were kept at 37°C, under an atmosphere containing 5% CO 2 in air. Cell viability was evaluated by the MTT assay, using a 24 h incubation protocol, according to a previously published procedure. 60 Two independent experiments were carried out, each comprising four replicate cultures.