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 BG, Italy). The melting points were determined on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and were 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 accumulating 64 scans with a 2 cm^–1 resolution in the region of 4000-650 cm^–1. All ¹H- and ¹³C-NMR spectra were recorded on a JEOL ECZR 400 MHz NMR spectrometer (400 MHz for ¹H and 100 MHz for ¹³C, Jeol, Tokyo, Japan) in dimethyl sulfoxide-d₆ (DMSO-d₆). ¹H and ¹³C chemical shifts (ẟ) 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™ Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo Scientific) equipped with a HESI II (heated electrospray ionization) source in the positive mode.

2-Hydroxynaphthalene-1-carboxylic acid (5.3 mmol) and the corresponding substituted aniline (5.3 mmol) were suspended in 30 mL of dry chlorobenzene. Phosphorous trichloride (2.65 mmol) was added dropwise, and the reacting mixture was heated in the microwave reactor for 15 min at 130 °C and maximal allowed power 500 W using infrared flask-surface control of temperature. The solvent was evaporated under reduced pressure, the solid residue was washed with 2 M HCl, and the crude product was recrystallized from aqueous ethanol. All the studied compounds are presented in Table 1.

2-Hydroxy-N-phenylnaphthalene-1-carboxamide (1), 2-hydroxy-N-(2-methoxyphenyl)naphthalene-1-carboxamide (2), 2-hydroxy-N-(3-methoxyphenyl)naphthalene-1-carboxamide (3), 2-hydroxy-N-(4-methoxyphenyl)naphthalene-1-carboxamide (4), 2-hydroxy-N-(2-methylphenyl)naphthalene-1-carboxamide (8), 2-hydroxy-N-(3--methylphenyl)naphthalene-1-carboxamide (9), 2-hydroxy-N-(4-methylphenyl)naphthalene-1-carboxamide (10), 2-hydroxy-N-(2-nitrophenyl)naphthalene-1-carboxamide (24), 2-hydroxy-N-(3-nitrophenyl)naphthalene-1-carboxamide (25), 2-hydroxy-N-(4-nitrophenyl)naphthalene-1-carboxamide (26) were described by Gonec et al. [43].

An HPLC system Agilent 1200 equipped with a DAD detector (Agilent, Santa Clara, CA, USA) was used. A chromatographic column Symmetry^® C₁₈ 5 μm, 4.6×250 mm, part No. WAT054275 (Waters Corp., Milford, MA, USA) was used. The HPLC separation process was monitored and evaluated with EZChrom Elite software ver. 3.3.2 (Agilent) [56]. Isocratic elution by a mixture of MeOH p.a. (72 %) and H₂O-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 p.a. (72 %) and acetate-buffered saline (pH 7.4 and pH 6.5) (28 %) as a mobile phase was used for the determination of distribution coefficients expressed as D_7.4 and D_6.5. The total flow of the column was 1.0 mL min^-1, the injection volume was 20 μL, the column temperature was 40 °C, and the sample temperature was 10 °C. The detection wavelength of 210 nm was chosen. A KI methanolic solution was used to determine 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 experimental values of lipophilicity of individual compounds are shown in Table 1.

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 [57] (Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Czech Republic), and MRSA SA 630 and MRSA SA 3202 [57] (National Institute of Public Health, Prague, Czech Republic), both of human origin. These three clinical isolates, carrying the mecA gene [58], were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 μg mL^-1 (VA2-MRSA) within the susceptible range for MRSA 63718) methicillin-resistant S. aureus (VS-MRSA) [57]. Vancomycin- and methicillin-susceptible S. aureus ATCC 29213 and vancomycin-susceptible Enterococcus faecalis ATCC 29212, obtained from the American Type Culture Collection, were used as the reference and quality control strains. Three vanA gene-carrying vancomycin-resistant isolates of E. faecalis (VRE 342B, VRE 368, VRE 725B) were provided by Oravcova et al. [59]. In addition, all the prepared compounds were tested against the Gram-negative bacteria E. coli ATCC 25922 (American Type Culture Collection).

The minimum inhibitory concentrations (MICs) were evaluated using the microtitration broth method according to the CLSI [60,61], with some modifications. The compounds were dissolved in DMSO (Sigma, St. Louis, MO, USA) to get a concentration 10 μg mL^-1 and diluted in a microtitration plate in an appropriate medium, i.e. Cation Adjusted Mueller–Hinton Broth (CaMH, Oxoid, Basingstoke, UK) for staphylococci, E. coli; and Brain Heart Infusion Broth (BHI, Oxoid) for enterococci to reach the final concentration of 256 to 0.125 μg mL^-1. Microtitre plates were inoculated with test microorganisms so that the final concentration of was 10⁵ bacterial cells in a microtiter plate. Ampicillin and ciprofloxacin (Sigma) were 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 all the tested bacteria. 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.

The evaluation of in vitro antimycobacterial activity of the compounds was performed against Mycobacterium tuberculosis ATCC 25177/H₃₇Ra, Mycobacterium kansasii DSM 44162 and Mycobacterium smegmatis ATCC 700084.

The broth dilution micro-method in Middlebrook 7H9 medium (Difco, Lawrence, KS, USA) supplemented with ADC Enrichment (Difco) was used to determine the minimum inhibitory concentration (MIC) as previously described [61]. The compounds were dissolved in DMSO (Sigma), and the final concentration of DMSO did not exceed 2.5 % of the total solution composition. The final concentrations of the evaluated compounds, ranging from 256 to 0.125 μg mL^-1, were obtained by twofold serial dilution of the stock solution in a microtiter plate with a sterile medium. Bacterial inocula were prepared by transferring colonies from culture to sterile water. The plate was inoculated by tested microorganisms. The final concentration of bacterial cells was 1.5×10⁶ for M. tuberculosis and 10⁵ cells in a microtiter plate for other mycobacteria. Isoniazid and rifampicin (Sigma) were used as reference antimycobacterial drugs. Drug-free controls, sterility controls, and controls consisting of medium and DMSO alone were included. The plates were incubated for a defined time at an appropriate temperature (3 days at 37 °C for M. smegmatis, and 14 days at 37 °C for M. tuberculosis and M. kansasii). After incubation, the MIC was visually evaluated as the lowest concentration of the tested compound, which completely inhibited the growth of the microorganism. The MICs against M. tuberculosis were evaluated by Alamar blue (Oxoid). After incubation, 10 % of Alamar blue was added to each well, and the plate was incubated for 24 h. The MIC values were assessed as the lowest concentration of the tested compounds, which prevented changing blue resazurin to pink resorufin. The experiments were repeated three times. The minimum inhibitory concentrations (MICs) were defined as the lowest concentration of the compound at which no visible bacterial growth was observed. The MIC value is routinely and widely used in bacterial assays and is a standard detection limit according to the CLSI [60]. The results are summarized in Table 3.

Cytotoxicity of the compounds was determined using an LDH assay kit (Roche Diagnostics, Mannheim, Germany) as described previously [43,54]. Human monocytic leukemia THP-1 cells (European Collection of Cell Cultures, Salisbury, UK) were exposed for 24 h at 37 °C to various compound concentrations ranging from 0.37 to 30 μM in RPMI 1640 medium. For LDH assays, cells were seeded into 96-well plates (5×10⁴ cells/well in 100 μL culture medium) in triplicate in serum-free RPMI 1640 medium and measurements were taken 24 h after the treatment with the compounds. The maximum concentration of DMSO (Sigma) in the assays never exceeded 0.1 %. Oxaliplatin and camptothecin (Sigma) were used as reference drugs. The median lethal dose values, LC₅₀, were deduced through the production of a dose-response curve. All data from three independent experiments were evaluated using GraphPad Prism 5.00 software (GraphPadSoftware, San Diego, CA, USA) [62]. The results are summarized in Table 3.

The compounds were prepared by a simple (click chemistry method) but innovative microwave synthesis from commercially available building blocks (2-hydroxy-1-naphthoic acid and multisubstituted anilines) in anhydrous chlorobenzene in the presence of PCl₃. The synthesis is depicted in Scheme 1 and a list of all studied compounds is given in Table 1. The unsubstituted derivative 1 and monosubstituted compounds 2-4, 8-10, 24-26 have already been described by Gonec et al. [43] but are listed here for the completeness of the entire study.

Since the basis for understanding the behavior of bioactive molecules is the knowledge of their lipo-hydrophilic properties, lipophilicity parameters were determined for all compounds, namely log k (logarithm of the capacity factor) and log D (logarithm of the distribution coefficient) at physiological pH 6.5 and 7.4.

The capacity factor and distribution coefficients were measured by RP-HPLC on a C18 column with methanol as an organic modifier of the mobile phase. In addition to the experimental lipophilicities, predicted log P values were also calculated using ACD/Percepta [55], see Table 1. In addition to lipophilicity, Table 1 also shows the predicted (ACD/Percepta [55]) electronic σ_(Ar) parameters of the whole substituted anilide ring, characterizing the electron-withdrawing or donating ability of the molecular system. The values of σ_(Ar) are found in a wide range from 0.01 to 1.36, so the compounds contain substituents with both electron-donating and slightly electron-withdrawing properties.

The graphs in Figure 1 show the correlations between the experimental and calculated lipophilicity values, and as can be seen from the correlation coefficients r, which range from 0.60 to 0.63 (n = 27), the agreement is small, which probably indicates a significant influence of the free phenol group, which the software cannot capture. On the other hand, the graphs in Figure 2 illustrate the relationships between log k and log D, which, according to the correlation coefficient r of ca. 0.99 (n = 27), are very good.

Figure 3 shows the order of the compounds according to the increasing log k value. The least lipophilic are methoxylated derivatives 7 (R = 3,4,5-OCH₃) and 4 (R = 4-OCH₃), while the most lipophilic are compounds 20 (R = 2-OCH₃-5-CF₃) and 19 (R = 2-OCH₃-5-Br). The unsubstituted derivative 1 (the fourth least lipophilic compound in this series) showed the largest deviation between the log k and log D values.

Regarding all these observations, it should be summarized that for these 2-hydroxynaphthalene-1-carboxamides on the ring-multisubstituted anilide, standard commercially available lipophilicity prediction programs are unable to provide relevant data due to the high incidence of intra- and intermolecular interactions. On the other hand, the presence of an ionizable acidic phenolic group in the vicinity of the amide bond does not cause significant differences in the experimental values obtained for different mobile phase properties/compositions.

The biological properties were assessed for in vitro antibacterial and antimycobacterial activity. In addition, all the compounds were also evaluated for their cytotoxicity against human monocytic leukemia THP-1 cells. The selection of the studied bacterial strains was adopted following the CLSI (National Committee for Clinical Laboratory Standards) international reference methodologies [63], i.e. standardization. For this purpose, universally sensitive collection strains from ATCC (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922) were selected. The second aspect of strain selection was the current state of occurrence of strains with an epidemiologically significant type of resistance, represented by clinical isolates of human and veterinary origin, i.e. different sequence types limited to human and animal populations, e.g. methicillin-resistant Staphylococcus aureus (MRSA) SA 3202, SA 630, 63718 isolates carrying the mecA gene [57]. In the case of vancomycin-resistant E. faecalis (VRE) 342B, 368, and 725B isolates carrying the vanA gene [59], these were isolates from wild birds colonized from US hospital wastewater, as confirmed. Therefore, it can be concluded that the tested strains differed in the spectrum of antibiotic resistance, genetic makeup, and probably accessory genome. Activities are expressed as minimum inhibitory concentrations (MICs), as shown in Table 2.

To obtain a comprehensive overview of the antibacterial properties of the investigated compounds, all derivatives were tested in vitro against Mycobacterium tuberculosis ATCC 25177/H₃₇Ra, Mycobacterium kansasii DSM 44162 and Mycobacterium smegmatis ATCC 700084; activities are expressed as MICs as reported in Table 3. In order to reduce risks, a replacement of model pathogens is commonly used in basic laboratory screening. For M. tuberculosis, avirulent strain H₃₇Ra is used, which has a similar pathology as M. tuberculosis strains infecting humans and, thus, represents a good model for testing antitubercular agents [64]. The genus Mycobacterium is a closely related group of fast- and slow-growing species. In addition to M. tuberculosis, there are a number of other so-called atypical (non-tuberculous) mycobacteria, important environmental pathogens, that cause a wide range of diseases (pulmonary diseases, lymphadenitis, skin and soft tissue diseases, gastrointestinal and skeletal infections), especially in immunocompromised patients [65-68]. These non-tuberculous strains include the fast-growing, e.g. M. smegmatis [69,70] and the slow-growing, e.g. M. kansasii [71,72].

In the early stage of the drug discovery process, especially orally administered drugs, it is extremely important to perform at least indicative ADME profiling to provide critical information about the basic behavior of a potential drug in the body. Basic information obtained from such studies helps guide further structural optimizations to obtain molecules with more favorable pharmacokinetic parameters while maintaining the existing efficiency potential. A variety of software is advantageously used for basic ADME screening, and ADME profiling is subsequently verified for selected drug candidates in preclinical and clinical studies [81-83]. The Lipinski Rule of Five (Ro5) is one of the most accepted recommendations concerning the physicochemical parameters of biologically active compounds, and all medicinal chemists try to follow it when designing molecules [84]. Ro5 contains the limits of specific molecular descriptors (MW <500, log P <5, HBD <5, HBA <10) set based on experimentally and statistically obtained results so that a compound that meets this recommendation has a higher chance of becoming a drug. However, a good drug-like score does not make a molecule a drug, and vice versa [85,86]. In addition, information has been added about compounds related to the Veber rule, which states that a compound with 10 or fewer rotational bonds (RB) and a polar surface area (TPSA) of no higher than 1.4 nm² (140 Ų) should have good oral bioavailability [81,87]. It is clear that ADMET-friendly properties, such as lipophilicity, polar surface area, etc, are important in the context of specific ligand-receptor interactions.

The following Table 4 shows the predicted ADME-influencing properties of the most effective compounds (13, 22, 27). The compounds are rather rigid and expected to be nearly planar [54]. All contain a free acidic phenolic group, which is crucial for biological activity [40,41,43], meaning that these acidic compounds in plasma bind predominantly to human serum albumin [88]. In addition to Ro5 parameters, other parameters, such as intestinal absorption and permeation into the brain (the effectiveness of antibiotics in the brain is important), are listed in Table 4. All the parameters were predicted using the commercially available program ACD/Percepta [55].

All the discussed agents have molecular weights (MW) significantly <500. On the other hand, the compounds have rather higher lipophilicity (log P ≥5). All the compounds meet the criteria for the number of H-bond donors (HBD) and acceptors (HBA). The number of rotatable bonds (RB) is in the narrow range of 2 to 4. The topological polar surface area (TPSA) has been recognized as a good indicator of intestinal drug absorption (TPSA <1.2-1.4 nm² (<120-140 Ų)) and blood-brain barrier (BBB) penetration (TPSA <0.6 nm² (<60 Ų)) [87,89,90]. The TPSA value of 0.49 nm² (49 Ų) indicates that compounds 13 and 22 should have good intestinal absorption as well as adequate BBB penetration. On the other hand, the TPSA = 0. 98 nm² (98 Ų) for compound 27 suggests only good intestinal absorption. The predicted value of the absorption rate in the jejunum (k_a = 0.055 min^-1) is the same for all derivatives. A remarkable prediction was made by ACD/Percepta [55] for blood-brain barrier (BBB) permeation. In general, log BB ≥ 0.3 is for BBB permeable drugs and log BB ≤ -0.3 is for impermeable drugs [91]. The log BB values of -0.11 (22) and -0.24 (27), respectively, indicate that both compounds are probably CNS inactive due to low brain penetration (as already suggested by the TPSA value for 27). Conversely, the log BB = 0.54 for 13 indicates good BBB penetration. More informative are the values of the permeability surface-area product (expressed as log PS) [92] in the range of -1.2 to 1.3. The brain plasma equilibrium rate predicted by ACD/Percepta [55] for compound 13 is -3.2, suggesting that this compound may achieve sufficient brain penetration for CNS activity.

In summary, after preliminary in silico ADME screening using commercially available software, it can be assumed that investigated compounds 13, 22, and 27 should have suitable physicochemical parameters for adequate bioavailability in the body. Unfortunately, for the most effective of the compounds 37, probably due to the presence of the NO₂ group, BBB penetration was not predicted. Of course, much more accurate results could be obtained using other experiments [93].