Novel Bis-Ammonium Salts of Pyridoxine: Synthesis and Antimicrobial Properties

Abstract

A series of 108 novel quaternary bis-ammonium pyridoxine derivatives carrying various substituents at the quaternary nitrogen's and acetal carbon was synthesized. Thirteen compounds exhibited antibacterial and antifungal activity (minimum inhibitory concentration (MIC) 0.25-16 µg/mL) comparable or superior than miramistin, benzalkonium chloride, and chlorhexidine. A strong correlation between the lipophilicity and antibacterial activity was found. The most active compounds had logP values in the range of 1-3, while compounds with logP > 6 and logP < 0 were almost inactive. All active compounds demonstrated cytotoxicity comparable with miramistin and chlorhexidine on HEK-293 cells and were three-fold less toxic when compared to benzalkonium chloride. The antibacterial activity of leading compound 5c₁₂ on biofilm-embedded Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli or Pseudomonas aeruginosa was comparable or even higher than that of the benzalkonium chloride. In vivo 5c₁₂was considerably less toxic (LD₅₀ 1705 mg/kg) than benzalkonium chloride, miramistine, and chlorhexidine at oral administration on CD-1 mice. An aqueous solution of 5c₁₂ (0.2%) was shown to be comparable to reference drugs efficiency on the rat's skin model. The molecular target of 5c₁₂ seems to be a cellular membrane as other quaternary ammonium salts. The obtained results make the described quaternary bis-ammonium pyridoxine derivatives promising and lead molecules in the development of the new antiseptics with a broad spectrum of antimicrobial activity.

Keywords: antibacterial activity; antifungal activity; antiseptics; biofilms; cytotoxicity; pyridoxine; quaternary ammonium salts.

Figure 1

The structures of QAC based on pyridoxine derivatives obtained by us earlier [13,14,15,16,17,18,19] and compounds 5a₈–5r₁₈ synthesized in this work.

Scheme 1

Reagents and conditions. (i) R₁C(O)R₂, HCl, 3–5 °C, 3 h; or C₆H₆, R₁C(O)R₂, p-TsOH, reflux, 17 h, (ii) H₂O_, NaOH, 20 °C, (iii) CH₂Cl₂, SOCl₂, 20 °C, 17 h, (iv) H₂O/CHCl_3, NaHCO₃, (v) CHCl₃, TCICA, reflux, 4 h, (vi) C₂H₅OH, R₃N(CH₃)₂, 70 °C, 5 h.

Figure 2

Lg1/MIC versus calculated logP relationship for novel bis-ammonium salts of pyridoxine. The parabolic line shows the polynomial approximation by using a second order model. Inactive compounds (MIC > 64 mg/mL) are not shown.

Figure 3

The effect of 5c₁₂ and reference antiseptics on biofilm-embedded bacteria. (A)—S. aureus; (B)—S. intermedius; (C)—E. coli, (D)—P. aeruginosa. The 48-h biofilms were treated with 1–16 × MBCs of benzalkonium chloride (circles), miramistin (squares), or 5c₁₂ (triangles) for 24 h and then quantified by CFU counting. Data are present as medians from five independent experiments with interquartile range (M ± IQR). Dotted line shows 3 log-decrease of viable cells.

Figure 4

In vivo antibacterial efficacy test (Mean ± SD) using a rat skin model. log10 number of viable cells in the control and the number of viable cells after exposure with 5c₁₂, miramistin, benzalkonium chloride, and chlorhexidine, n = 6.

Figure 5

The effect of 5c₁₂ on the electric potential of the bacterial membrane determined using a membrane-potential-sensitive cyanine dye DiSC₃(5) (10 min treatment by 1–8 × MBC (A,B) and 20 min treatment by 1–8 × MBC (C,D)). Asterisks denote a statistically significant difference in comparison with untreated cells. * statistically significant difference from the control.

Figure 6

Scanning Electron Microscopy microphotographs of S. aureus ATCC209p (A,C) and E. coli CDC F-50 (B,D). Untreated cells (A,B) and bacteria treated by 0.2% solution of 5c₁₂ for 30 min (C,D). 50,000× magnification.