Synthesis and Antimicrobial Activity of Carboxylate Phosphabetaines Derivatives with Alkyl Chains of Various Lengths

e purpose of the present study was to investigate the antibacterial activity of ��een novel nanosized alkyl esters of carboxylate phosphabetaine: ββ-(carboxyalkyl)ethyltriphenylphosphonium bromides 4–8, ββ-(carboxyalkyl)-ββmethylethyltriphenylphosphonium bromides 9–13, and ββ-(carboxyalkyl)-αα-methylethyltriphenylphosphonium bromides 14–18. e in vitro microbiological activity of the synthesized phosphonium bromides against gram-positive and gram-negative bacteria and the yeast Candida albicans was determined in comparison to standard agents. Microbiological results indicate that the synthesized phosphonium salts 4–18 possess a broad spectrum of activity against the tested microorganisms. Every newly synthesized compound was characterized by elemental analyses, IR, HNMR, and P NMR spectral studies.


Introduction
Demand for new antimicrobial agents is high because more microorganisms develop resistance against drugs currently available on the market.Resistance of pathogenic bacteria to antibiotics is rapidly becoming a major problem in the medical community and hospital-based healthcare settings.e search for novel agents to combat resistant bacteria has become one of the most important areas of antibacterial research today [1,2].Pharmaceutical and organic chemists are trying to synthesize new drugs with better pharmacokinetic and dynamic properties.
In this study, we prepared triphenyl-substituted phosphonium salts 4-18 on the base of phosphabetaines (1)(2)(3) containing alkyl chains of various lengths.e synthesis of such phosphonium salts is very difficult in comparison with ammonium analogs [3,4].In the past years, our group carried out regular research on the synthesis, structure, and reactivity of phosphabetaines of type 1-3, obtained on the basis of tertiary phosphines and unsaturated carboxylic acids [5][6][7][8].
e surging interest in this class of compounds becomes quite understandable if we take into account the fact that phosphabetaines are also the original analogs of organic amino acids, with a wide spectrum of potential chemical and biological properties.In these internal phosphonium salts, cationic phosphonium and anionic centers are interconnected not only by ionic but also by covalent bonds.e structure of all products 1-3 has been con�rmed by the direct X-ray method [6][7][8].

Experimental
2.1.Chemistry.All materials were obtained from commercial suppliers and used without puri�cation.Analytical data were obtained from Perkin Elmer 2400 LS and were found within ±0.4% of the theoretical values.Infrared (IR) spectra were recorded on using KBr disk on Specord M-80. 1 H NMR (D 2 O) and 31 P NMR (DMSO-d 6 ) spectra were determined on a Bruker Avance digital spectrometer 400 MHz.

Synthesis of 𝛽𝛽-(Carboxyalkyl)ethyltriphenylphosphonium
Bromides (4-8)   2.5.Antimicrobial Screening.e antimicrobial activity of the newly synthesized compounds was determined in vitro using the agar disk-diffusion method using Mueller-Hilton agar medium [9,10] against a variety of pathogenic microorganisms: Staphylococcus aureus (ATCC 29213) (Gram-positive bacteria), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Proteus mirabilis (ATCC 12453) (Gram-negative bacteria) and fungus Candida albicans (ATCC 885-653).e inhibition zones of the tested compounds were measured aer 24-48 h incubation at 37 ○ C for bacteria and aer 5 days of incubation at 28 ○ C for fungi.Penicillin (Sigma-Aldrich) and Chlorhexidine (Sigma-Aldrich) were used as reference drug for bacteria, whereas Griseofulvin (Sigma-Aldrich) was used as reference drug for fungi.Every experiment in the antibacterial and antifungal assay was replicated twice.For the antibacterial and antifungal activity, the compounds were dissolved in dimethylsulfoxide (DMSO).Many years we thought that it is impossible to grow single crystals of oil products 4-18 with long alkyl chains suitable for X-ray diffraction, but aer �ve years we have a real chance to obtain the crystalline structure of the Ph 3 PCH 2 CH 2 C(O)OC 16 H 33 (7), which gave good quality crystals (Figure 1) [11].

Antibacterial Activity.
Different strains of bacteria were used as Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Proteus mirabilis (ATCC 12453).Cup-plate Agar method was used for evaluation of antibacterial activity.e nutrient agar medium is used.e medium with bacteria was poured into sterilized Petri dishes under aseptic conditions.Standard drugs used were Penicillin (50 g/0.1 mL) and Chlorhexidine (50 g/0.1 mL) and test compounds at concentration of 50 g/0.1 mL.Solvent used was dimethyl sulfoxide (DMSO).Plates were incubated at 37 ○ C for 24 hours.Aer incubation the average zone of inhibition was recorded in mm [12].
2.5.2.Antifungal Activity.e antifungal activity was carried out by using cup-plate method using Sabouraud's agar medium.Fungal strains used were Candida albicans (ATCC 885-653) with incubation period of 48 hours at temperature 28 ○ C. e standard drug used was Griseofulvin (50 g/0.1 mL) and the test compounds at concentration of 50 g/0.1 mL by using dimethyl sulfoxide (DMSO) [13].

Results and Discussions
3.1.Chemistry.e synthetic routes are given in Schemes 2-4.In this paper we present the synthesis and biological activity of a series of nanosized (30 nm) quaternary phosphonium salts 4-18 with long alkyl chains (R=C  H 2+1 ;  = 10, 12, 14, 16, 18; here  is the number of carbon atoms in alkyl groups) on the basis of phosphabetaines 1-3 and higher alkyl halogenides.All the synthesized compounds were characterized by elemental analysis, IR, 1 H NMR, and 31 P NMR spectroscopy.A crystalline product of -(carboxyhexadecyl)ethyltriphenylphosphonium bromide 7 was prepared and characterized by single-crystal X-ray analysis [11] (Figure 1).

Scheme 2 Depicts the Synthesis of 𝛽𝛽-(Carboxyalkyl)
ethyltriphenylphosphonium Bromides (4)(5)(6)(7)(8).Treatment of acrylic acid with triphenylphosphine at room temperature in chloroform during 6 hours yielded (78%) phosphabetaine 1. Alkylation of the starting phosphabetaine 1--triphenylphosphonium ethylcarboxylate with alkyl halogenides (re�ux for two hours in CH 3 Cl) gave the corresponding phosphonium bromides 4-8 with long alkyl chains.Molecular structure of product 7 is given in Figure 1.  1.Such a high biological activity of cationic biocides 4-18 we explain by their ability to be integrated into the lipid layers of biomembranes of pathogenic micro�ora eventually leading to the destruction of this last [14].To con�rm this idea we studied the interaction mechanism of compounds 4-8-synthetic phosphorus analogs of biomembranes-with natural biological membranes (lecithin) using the model of Langmuir monolayers [15].It was discovered that alkylated phosphabetaines 4-8 interact with lecithin, by forming a pores, and thus deteriorating the membrane functions.

Conclusion
In conclusion, carboxylate phosphabetaines derivatives with alkyl chains of various lengths were synthesized in good yield, characterized by different spectral studies, and their antimicrobial activity has been evaluated.Compounds 5-8, 11, 15, and 16 demonstrated good inhibitions against all the strains tested comparable to Chlorhexidine, Penicillin, and Griseofulvin as positive standard.So, it may be concluded from our results that the synthesized compounds are potent nanoantimicrobial agents against pathogenic bacteria and fungi.