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

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.

Antibacterial activity profile of miramistin in in vitro and in vivo models.

BACKGROUND: Miramistin is a widely used antiseptic, disinfectant and preservative, and one of the most popular antimicrobial agents on pharmaceutical market of the Russian Federation (http://www.dsm.ru/en/news/385/). However, there is a lack of reported systematic data on antibacterial efficacy of this agent obtained in accordance with the international standards. AIM: This paper represents a systematic study of antibacterial properties of miramistin. Another objective of this work is to evaluate and compare the exploratory performance of in vitro and in vivo protocols of antiseptics' efficacy testing using miramistin as the reference antiseptic. METHODS: Antibacterial activity of 0.1% and 0.2% aqueous solutions of miramistin against two museum strains of S. aureus (ATCC 209p) and E. coli (CDC F-50) was studied. Three standard in vitro laboratory tests (microdilution test, suspension test, and metal surface test), and one in vivo test (on rat's skin) were used. The study was conducted in accordance with the international regulatory documents. RESULTS: Miramistin showed high bactericidal activity against the studied bacterial pathogens in the standard in vitro tests. Thus, in the microdilution test it showed expressed activity against S. aureus (MIC 8 µg/ml, MBC 16 µg/ml) and E. coli (MIC 32 µg/ml, MBC 128 µg/ml). In the suspension test, miramistin decreased the amount of colony forming units by at least 6 log10 units for S. aureus, and by at least 4.5 log10 units for E. coli. Transition to the metal surface test led to significant decrease of antibacterial activity by 1-3 log10 units as compared to the suspension test. Further dramatic reduction of antiseptic activity (by 3-4 log10 units) was observed in in vivo rat skin test. Addition of a protein contaminant (bovine serum albumin) led to a general decrease in the effectiveness of miramistin against the test pathogens (typically, by 1-2 log10 units). An interesting effect of exposure time-dependent reversal of miramistin's specificity to the studied Gram-positive S. aureus and the Gram-negative E. coli organisms was observed in the metal surface test. CONCLUSIONS: The results of this work provide systematic data on antibacterial efficacy of miramistin. They also underscore the need in relevant in vivo models for evaluation of antiseptics' efficacy. While the existing in vitro methods can be successfully applied at the discovery stages, it is necessary to use more realistic in vivo models at more advanced development stages. The observed selectivity reversal effect should be taken into account when carrying out the antiseptics' efficacy testing and surface disinfection procedures.

Antibacterial effects of quaternary bis-phosphonium and ammonium salts of pyridoxine on Staphylococcus aureus cells: A single base hitting two distinct targets?

We studied the effects of quaternary bis-phosphonium and bis-ammonium salts of pyridoxine with lipophilic substituents on the survival and morphology of Staphylococcus aureus cells. We found that, while originating from the same base, they exhibit considerably different antimicrobial mechanisms. In the presence of Ca(2+) ions the MIC and MBC values of ammonium salt increased 100-fold, suggesting that Ca(2+) ions can successfully impede the membrane Ca(2+) ions exchange required for ammonium salt incorporation. In contrast, in the presence of quaternary phosphonium salt, the artificial capsular-like material was formed around the cells and the filamentous and chain-like growth of the cells was observed suggesting the disruption of the cell division mechanisms. Altogether, both pyridoxine derivatives successfully inhibited the growth of gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis) and Escherichia coli considerably, while demonstrated nearly no effect against Klebsiella pneumoniae and Pseudomonas aeruginosa. We suggest that due to their effects on distinct and likely complementary targets the derivatives of pyridoxine represent potentially perspective antibacterials with complicated adaptation and thus with lower risk of drug resistance development.

Synthesis and Antibacterial Activity of Novel Quaternary Ammonium Pyridoxine Derivatives.

A series of 26 quaternary ammonium pyridoxine derivatives were synthesized and their cytotoxicity and antibacterial activities against clinically relevant bacterial strains were tested in vitro. The antibacterial activity of mono-ammonium salts increased with the rise of the lipophilicity and compound 3,3,5-trimethyl-8,8-dioctyl-1,7,8,9-tetrahydro-[1,3]dioxino[5,4-d]pyrrolo[3,4-b]pyridin-8-ium chloride (2d) reaches a maximum among them. Bis-ammonium salt of pyridoxine 4 with two dimethyloctylamine groups also demonstrated high antibacterial activity despite lower lipophilicity. The results of MTT assay indicated that HEK 293 cells were more sensitive than HSF to quaternary ammonium pyridoxine derivatives. Compounds 2d and 4 did not induce the damage of the DNA and might be of interest in the development of new antimicrobials.