In Vitro Antibacterial and Antifungal Activity of Salicylanilide Benzoates

The resistance to antimicrobial agents brings a need of novel antimicrobial agents. We have synthesized and found the in vitro antibacterial activity of salicylanilide esters with benzoic acid (2-(phenylcarbamoyl)phenyl benzoates) in micromolar range. They were evaluated in vitro for the activity against eight fungal and eight bacterial species. All derivatives showed a significant antibacterial activity against Gram-positive strains with minimum inhibitory concentrations ≥0.98 μmol/L including methicillin-resistant Staphylococcus aureus strain. The most active compounds were 5-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl benzoate and 4-chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl benzoate. The antifungal activity is significantly lower.


Introduction
The worldwide epidemic of antibiotic resistance is in danger of ending the "golden age" of antibiotic therapy and therefore is touching all people [1]. Major current problems arise from the spread of nosocomial antibioticresistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamases-producing (ESBL) Escherichia coli or Klebsiella spp., multiresistant Pseudomonas, or Acinetobacter sp. as well as Clostridium difficile [2].
The situation in the fungal kingdom is a bit different. Over the past decades there has been a growing number of immunocompromised patients (e.g., patients with AIDS or after transplantations) who can develop opportunistic mycoses caused by expanding spectrum of fungal pathogens, including those with problematic susceptibility to current antifungal drugs. Pathogenic fungi can use different mechanisms of resistance to diverse drugs with unrelated modes of action [3].
The searching for potential antimicrobial agents is still challenging and new groups of compounds are desired [4].
Various salicylanilide (2-hydroxy-N-phenylbenzamide) esters have displayed good antibacterial and antifungal activities, especially against Gram-positive strains [5][6][7]. Recently salicylanilides were described besides an excellent antibacterial acting against both drug-sensitive and methicillinresistant S. aureus inhibition activity towards bacterial transglycosylase, an enzyme necessary for the formation of the cell wall [8].
Benzoic acid alone is known as a nonspecific antimicrobial agent with the wide spectrum of the activities against human pathogenic fungi and bacteria with different minimum inhibitory concentration (MIC) values [9][10][11][12][13][14]; moreover it was being evaluated as an inhibitor of β-carbonic anhydrase, a new molecular target occurring in C. albicans and Cryptococcus neoformans [15]. The review of the benzoic acid as preservative agent, the mechanisms of action, and resistance were published [16]. 2 The Scientific World Journal Based on these facts, we designed and evaluated new salicylanilide benzoates as potential antibacterial and antifungal agents.

Material and Methods
2.1. Chemistry. Salicylanilides were prepared by the procedure described previously [7]. The esters were prepared from salicylanilides by using benzoic acid and N,N -dicyclohexylcarbodiimide as dehydrating and condensation agent (e.g., [7]). The general structure is presented in the head of Table 1.
All used chemicals were purchased from commercial sources (Sigma-Aldrich) and they were used without a further purification. Reactions were monitored by thin-layer chromatography plates coated with 0.2 mm silica gel 60 F 254 (Merck) visualized by UV irradiation (254 nm). All synthesized compounds were characterized. Elemental analysis (C, H, N) was performed on an automatic microanalyser CHNS-O CE instrument (FISONS EA 1110, Italy). Melting points were determined on a Melting Point machine B-540 (Büchi) apparatus using open capillaries and they are uncorrected. Infrared spectra (ATR) were recorded on FT-IR spectrometer Nicolet 6700 FT-IR in the range of 400-4000 cm −1 . The NMR spectra were recorded on a Varian VNMR S500 (500 MHz for 1 H and 125 MHz for 13 C; Varian, Inc., Palo Alto, USA) at ambient temperature using deuterated dimethyl sulfoxide (DMSO-d6) solutions of the samples. The chemical shifts δ are given in ppm, with respect to tetramethylsilane as an internal standard. The coupling constants (J) are reported in Hz. The microdilution broth method modified according to standard M07-A07 [17] in Mueller-Hinton broth (HiMedia Laboratories, India) was adjusted to pH 7.4 (±0.2). The investigated compounds were dissolved in DMSO to the final concentrations ranging from 500 to 0.49 μmol/L. Penicillin G (benzylpenicillin) and benzoic acid were used as comparative standard drugs. Bacterial inoculum in sterile water was prepared to match 0.5 McFarland scale (1.5 × 10 8 CFU/mL). The minimum inhibitory concentrations (MICs) were assayed as 80% (IC 80 ) or higher reduction of growth in comparison to the control. The determination of results was performed visually and spectrophotometrically (at 540 nm). The values of MICs were determined after 24 and 48 h of incubation in the darkness at 35 • C (±0.1) in a humid atmosphere.
The method used was microdilution broth method in the format of the CLSI M27-A3 and M38 A2 guidelines for yeasts and moulds [18,19] in RPMI 1640 with glutamine (KlinLab, the Czech Republic) buffered to pH 7.0 with 0.165 M of 3-morpholino-propane-1-sulphonic acid (Sigma-Aldrich, Germany). DMSO served as a diluent for all compounds. Fungal inoculum was prepared to give a final concentration of 5 × 10 3 ± 0.2 CFU/mL. Fluconazole was used as a reference drug. Other conditions were the same as for antibacterial assay; only for T. mentagrophytes the final MIC were determined after 72 and 120 h of incubation. MICs were determined twice and in duplicate.   (14).   3H, m, H3 , H4 , H5 ). 13 8, 12, 17) and other worse than benzylpenicillin.
Gram-negative species (E. coli, two strains of Klebsiella pneumoniae, P. aeruginosa) were almost completely resistant to benzoates up to 125 μmol/L at the testing conditions with one notable exception; P. aeruginosa was inhibited by 1 at low concentration of 3.9/7.81 μmol/L.

Antifungal
Evaluation. Synthesized esters were tested for their in vitro activity against eight human pathogenic fungi. MIC values are presented in Table 2. Benzoic acid alone was completely inactive at the concentration of 500 μmol/L at both neutral and slightly acidic (pH ∼ 5) environment.
Unforeseen, in contrast to the antibacterial activity, salicylanilide benzoates expressed only mild antifungal potency. From eight strains, C. tropicalis, C. glabrata, and A. fumigatus showed a complete insensitivity to all tested derivatives at the value of 125 μmol/L. When concentrated on the esters, benzoates 2 and 11 did not affect the growth of any fungal species at the concentration of 500 μmol/L and lower, derivatives 12, 14, and 17 up to 250 μmol/L and 3, 4, 5, 7, 10, 15, 16 expressed activity levels >125 μmol/L. Only six derivatives (1, 6, 8, 9, 13, 18) displayed certain in vitro efficacy. The most active compound was assayed trichlorinated ester 6 (MIC ≥ 3.9 μmol/L), which surpassed standard fluconazole against C. krusei. In general, Candida is more resistant to salicylanilide esters than moulds and moreover, it seems that the mechanism of the action is only fungistatic. On the other side, the growth of T. mentagrophytes was affected by the highest number of the derivatives; based on the MIC values, activity against filamentous fungi is probably fungicidal.
Because pH effect on the efficiency of benzoic acid derivatives (generally of weak organic acids) was described (e.g., [16]), we measured MIC values of the esters 6 and 15 not only at approximately neutral environment, but additionally at slightly acidic pH (∼5; without buffering of the testing medium). Unfortunately, the change of pH did not result in the improvement of activity, even it led to a bit worse values.
Although we expected that the introduction of benzoyl fragment into salicylanilide molecules resulted in the significantly increased antifungal potency, this modification failed in this point.

Conclusion
In sum, we have designed and synthesized new esters of halogenated salicylanilides with benzoic acid. This series of compounds was evaluated to be a new group with promising in vitro antibacterial (against Gram-positive strains) activity. Unfortunately these derivatives disappointed expectation about them as potential antifungal agents.