Dibasic Derivatives of Phenylcarbamic Acid as Prospective Antibacterial Agents Interacting with Cytoplasmic Membrane

Abstract

1-[2-[({[2-/3-(Alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium/azepan- ium oxalates or dichlorides (alkoxy = butoxy to heptyloxy) were recently described as very promising antimycobacterial agents. These compounds were tested in vitro against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 (reference and control strains), three methicillin-resistant isolates of S. aureus, and three isolates of vancomycin-resistant E. faecalis. 1-[3-(Dipropylammonio)-2-({[3-(pentyloxy-/hexyloxy-/heptyloxy)phenyl]carbamoyl}oxy)propyl]pyrrolidinium dichlorides showed high activity against staphylococci and enterococci comparable with or higher than that of used controls (clinically used antibiotics and antiseptics). The screening of the cytotoxicity of the compounds as well as the used controls was performed using human monocytic leukemia cells. IC50 values of the most effective compounds ranged from ca. 3.5 to 6.3 µM, thus, it can be stated that the antimicrobial effect is closely connected with their cytotoxicity. The antibacterial activity is based on the surface activity of the compounds that are influenced by the length of their alkoxy side chain, the size of the azacyclic system, and hydro-lipophilic properties, as proven by in vitro experiments and chemometric principal component analyses. Synergistic studies showed the increased activity of oxacillin, gentamicin, and vancomycin, which could be explained by the direct activity of the compounds against the bacterial cell wall. All these compounds demonstrate excellent antibiofilm activity, when they inhibit and disrupt the biofilm of S. aureus in concentrations close to minimum inhibitory concentrations against planktonic cells. Expected interactions of the compounds with the cytoplasmic membrane are proven by in vitro crystal violet uptake assays.

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“…Antimicrobial activity of compounds was tested using microtitration broth method according to Clinical and Laboratory Standards Institute [44] with some modifications described previously [45][46][47]. Compounds were dissolved in DMSO to get concentration 10 mg/mL and diluted in a microtitration plate in an appropriate medium, to reach the final concentration 256-2 µg/mL.…”

“…The absorbance at 595 nm was measured (Tecan Infinite 200 PRO, Tecan, Grodig, Austria) and the percentage of inhibition was determined. As a blank, a non-inoculated plate treated in the same way was used [46,48]. The ability to inhibit biofilm formation was evaluated as a percentage inhibition of growth compared to the growth control according to the equation: % of inhibition = 100 − (OD 595S /OD 595C ) × 100 where OD 595S is the absorbance of the sample at 595 nm, and OD 595C is the absorbance of the growth control at 595 nm.…”

“…The solution in the wells was removed, and the plates were washed with PBS. The formazan crystals were dissolved by using 17% sodium dodecyl sulphate in 40% dimethylformamide [46,49]. The absorbance at 570 nm was measured and the percentage of eradication was determined according to the equation: % of biofilm eradication = 100 − (OD 570S /OD 570C ) × 100 where OD 570S is the absorbance of the sample at 570 nm, and OD 570C is the absorbance of the growth control at 570 nm.…”