Synthesized Phosphonium Compounds Demonstrate Resistant Modulatory and Antibiofilm Formation Activities against Some Pathogenic Bacteria

Department of Pharmaceutical Chemistry, Drug Discovery Unit, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana Analytical Laboratory, Ghana Standards Authority, Accra, Ghana Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana Department of Pharmaceutics, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana


Introduction
Infectious diseases have over the years threatened the survival of humans globally [1]. As one of the leading causes of mortality, infectious diseases caused by parasites, bacteria, viruses, and fungi result in over one-fourth of deaths worldwide every year [1,2]. Since these pathogens are ubiquitous and can spread through the air, water, and food, their control and subsequent prevention continue to be a daunting task. To combat these microorganisms, all kinds of antimicrobial agents including antibiotics, disinfectants, and antiseptics have been used [3]. However, the irrational or indiscriminate use of most antibiotics has triggered the emergence of resistant strains of microbes, leading to slow progress in the fight against these pathogens. is continuous insurgence of resistance strains calls for the search for compounds or antibacterial agents with new mechanisms of action. Presently, quaternary ammonium salts, which are positively charged compounds, mostly found in disinfectants have been widely used as antibacterial agents [4]. On the other hand, quaternary phosphonium salts are positively charged lipophilic cations with established antibacterial activities and a better killing rate than their quaternary ammonium counterparts but have been underutilized in pharmaceutical formulations though they can better enhance the antimicrobial efficacy [5]. For instance, various phosphonium salts possessing alkyl chains of different carbon lengths exhibited antimicrobial activities [6]. e linking of different functionalities together has been one of the tools in designing drugs to overcome antimicrobial resistance [7,8]. is is evident in some hybrid compounds containing a heterocyclic ring and phosphonium salts showing antimicrobial activity [9].
Hence, this study sought to synthesize some hybrid phosphonium salts with potential antimicrobial activity against human pathogens.

Chemistry
2.1.1. General. All solvents and reagents for the synthesis of the target compounds were obtained from commercially available sources including, among others, Sigma-Aldrich, Fisher Scientific, Fluorochem, and Alfa Aesar. Solvents were used directly in the reaction flask or as anhydrous via the nitrogen or argon line. in-layer-chromatography (TLC) analysis was performed on silica gel plates (E. Merck silica gel 60 F254 plates) and visualized by ultraviolet (UV) radiation at 254 nm. e compounds were purified by gravity column chromatography with silica gel as a stationary phase (Merck 60, 230−400 mesh). Infrared (IR) spectra were recorded in the range 400-4000 cm −1 using a Perkin-Elmer FT-IR spectrophotometer (Japan) which employed attenuated total reflection (ATR) with internal calibration. Samples were run neat. 1 H, 13 C nuclear magnetic resonance (NMR) and DEPT-135 analysis were performed on a Bruker Ascend FT NMR 500 MHz spectrometer (NM 103508-10, Germany) using TMS (residual solvent) as the internal standard and deuterated dimethyl sulphoxide (DMSO-d6) to prepare the samples and as the lock sourced from Cambridge Isotope Laboratories Inc (United Kingdom). 31 P spectra were also obtained for the phosphonium salts after employing a phosphorous probe. Spectroscopic data was assigned with the following abbreviations: br-broad, s-singlet, d-doublet, t-triplet, and m-multiplet. Chemical shifts were recorded in ppm. Coupling constants J are expressed in Hz. High-Resolution Mass spectra (MS) were measured on 6420 Triple Quad with an electrospray ionization source from Agilent Technologies. e purity of all tested compounds was determined by analytical RP-HPLC in tandem with MS using Agilent 1290 infinity series and a Mightysil C18 column (150 mm × 4.6 mm, 15 μm) using a gradient elution system and a flow rate of 1 ml/min at photodiode array detection wavelength at 270 nm. Mass spectra were registered in both ESI (−ve) modes. Uncorrected melting points were determined by an open capillary method using a Gallenkamp (C9759, England) melting point apparatus. All the compounds tested for their biological activity are >90% pure, confirmed with 1 H NMR and HPLC-MS. Scanned spectra for all compounds are shown in Figures SM5-SM25.

General Procedure 1: Azides' Formation.
e synthesis of azides in Scheme 3.1 as described by Bock and coworkers was followed [10]. is scheme was applied to the synthesis of nitroaromatic azides. e azides were used without further purification for the 'click' reactions.

General Procedure 3: Bromination of the 1,2,3-Triazoles.
e bromination process in Scheme 3.4 as described by Zhao and coworkers was followed, replacing the starting material with the 'clicked' product in general procedure 2 [12].

General Procedure 4: Synthesis of the Phosphonium Salts.
e phosphonium salts synthetic procedure as described by [13] was followed. e scheme for the synthesis of the phosphonium salts and a tethered/hybrid phosphonium salt is shown in Figure 1.

Determination of Minimum Inhibitory Concentration (MIC).
e minimum inhibitory concentrations (MICs) for each compound against the Gram-positive, S. aureus (ATCC 25923) and E. faecalis (ATCC 29212); Gram-negative, P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922)); and Mycobacterium smegmatis mc 2 155 isolates were determined using the high-throughput spot culture growth inhibition (HT-SPOTi) assay. Stock solution of the compounds P-MEOXY (1), 2ET-TOL (2), 2ET-FLU (3), RABYL-TOL (4), PPh3Br (5), and P-Z (6) (50 mg/mL) were prepared in DMSO followed by a twofold serial dilution in a 96-well halfskirted plate to give a concentration range of (50-0.05 mg/ mL). Subsequently, 2 µL of each dilution was dispensed into the corresponding wells of the 96-well plate followed by the addition of 200 μL of nutrient agar/Middlebrooks 7H10 agar with shaking. e final concentration range in the agar ranged between 500 and −0.5 μg/mL. e wells were then spotted with 2 µL of cell suspension (∼1 × 10 6 CFU/mL) using a multichannel pipette. Column 12 of the 96-well plate with agar and cells only were used as positive control. e plates were wrapped in aluminum foil and incubated at 37°C for 24 h. e MIC was defined as the lowest compound concentration that completely inhibited the growth of the microorganisms.

Modulation of Antibiotic Resistance Studies.
To evaluate the ability of the compounds to modulate antibiotic resistance, the combinatory effects of the most effective compounds (P-MEOXY (1), 2-ET-TOL (2), and RABYL-TOL) (4) were evaluated with the antibiotic amoxicillin against P. aeruginosa and S. aureus. e checkerboard distribution method in a 96-well microtitre plate was employed using the HT-SPOT inhibition assay. e fractional inhibition concentration index was calculated using the equation below. It was interpreted as FICI: ≤ 0.5, synergy; > 0.5 ≤ 4.0, no interaction; >4.0, antagonism. (1)

Biofilm Inhibition Studies.
e ability of the compounds to inhibit biofilm formation was evaluated using the microplate-based assay and the bacterial cells were cultured in tryptic soy broth (TSB) at 37°C overnight. e cultures were then harvested and resuspended in TSB to approximately 1 × 10 6 cfu/ml determined by the optical density (OD). Twofold serial dilution of the compounds was carried out with tryptic soy broth (TSB) to obtain concentrations ranging from 1/2 to 1/16 MIC. e compounds (10 μL) each were then premixed with the bacterial inoculum (190 μL). After 24 h incubation at 37°C, the supernatant containing TSB and planktonic cells was gently removed from the microplate wells, and the wells were washed twice with 150 μL of phosphatebuffered saline (PBS). After rinsing, the biofilms were heat-fixed by incubating at 60°C for 60 minutes. Biofilms were stained with 125 μL of 0.01% (w/v) crystal violet solution, and the suspension was incubated for 1 h at 37°C. Excess crystal violet solution was rinsed with distilled water and dried at room temperature for 30 minutes. After drying the stained plates, biofilms were visible as purple rings on the sides of each well. e quantitative analysis of biofilm formation was carried out by adding 125 mL of 95% v/v ethanol and acetic acid (3 : 1) per well. e OD of the stain was then measured at 600 nm using a multimode microtitre plate reader. Wells containing bacteria and TSB and wells containing only TSB were included as negative control and media control, respectively. e bioassay was performed in triplicate. e inhibitory activity was expressed as percentage biofilm inhibition using the equation: Check the font size and reduce it a little for the formula so that it is a bit uniformed.

Statistical Analysis.
e results obtained from the study were analyzed using GraphPad Prism 6.0 software (Version 8.01, GraphPad Software Inc., USA). e analysis of variance (ANOVA) was followed by the Neuman-Keuls posttest. All the values are expressed as mean ± standard error of the mean (SEM) from triplicate experiments.

Chemistry.
e synthesis centered on nonclicked and hybrid (clicked) compounds bearing lipophilic groups ( Figure 2) due to evidence from literature about the relationship between the lipophilic character of compounds and their biological activities. Optimization of the structural scaffold possibly enhances the biological activity with different electronic donating/withdrawing groups such as methyl, methoxy, and fluoro on the phosphonium head. In the first attempt to synthesize the nonclicked compounds (1, 2, and 5), a reaction was carried out between benzyl bromide and various triphenylphosphines in toluene under reflux conditions. e reaction afforded precipitates that were crushed out of the solution and isolated by suction filtration under vacuum as white solids. In the synthesis of the clicked compounds (2, 3, and 6), the products did not require further purification and were characterized by spectroscopy (NMR, FT-IR, HRESIMS). e synthesis of the clicked compounds involved the first step of generating p-nitrobenzyl azide from p-nitrobenzyl bromide in water: acetone (1 : 4) mixture at room temperature for 6 hrs.
is was followed by the copper-catalyzed 1, 3-dipolar cycloaddition in an organoaqueous solvent mixture at room temperature for 8 hrs. e reaction was monitored by thin layer chromatography (TLC). e product was worked up using diethyl ether in a solvent-solvent extraction and the combined organic portions were concentrated in vacuo using a rotary evaporator to obtain a beige solid which was further purified by silica column chromatography in a gradient elution fashion to afford a white solid hereafter called the clicked compound. After confirming the structure of the clicked compound by spectroscopic characterization, the clicked compound was reacted with phosphorous tribromide to convert the hydroxy group to a good leaving group, bromine.
is made the brominated product susceptible to nucleophilic substitution bimolecular reaction (SN 2 ) reaction with respective triphenylphosphines to introduce the substituted phosphonium heads to form the hybrid compounds. ey were characterized by spectroscopy (NMR ( 1 H, 13 C/DEPT-135, and 31 P), FT-IR, HRE-SIMS). e synthetic scheme is represented in Figure 1 and the supplementary information is in Figures SM1-SM25. e synthesis of the library of phosphonium salts (1-6) was performed according to methods in literature appended to the various procedures. e compounds were obtained with good yields and high purity confirmed by melting point and spectroscopic methods. e treatment of nitro benzyl bromides with various triphenylphosphine derivatives afforded compounds 1, 4, and 5. via a nucleophilic substitution bimolecular reaction. Copper-catalyzed azide-alkyne 1, 3-dipolar cycloaddition click reaction was used to attach the 1,2,3-triazole intermediate to the phosphonium salts as the hybrids (2, 3, and 6) after creating the brominated functionality which provided the pathway for the final step via nucleophilic substitution bimolecular (SN 2 ) mechanism. e structures of the synthesized compounds 3-6 were established on the basis of IR, NMR ( 1 H, 13 C, and DEPT-135), and LC-MS spectroscopic data, and thereafter, IR and 1 H NMR were used to confirm the nonhybrids (compounds 1-3). For instance, the FT-IR spectrum for detecting the functional groups of  Figures SM11-SM15).

Susceptibility of Compounds to Test Organisms in Minimum Inhibitory Concentration (MIC) Determinations.
e potency of the test compounds to inhibit bacterial growth was quantitatively assessed by the MIC values obtained experimentally (Table 1). Five different pathogens were used, of which two were Gram-negative bacteria, two were Gram-positive bacteria, and an acid-fast bacterium. Four of the test organisms showed susceptibility to the test compounds while Enterococcus faecalis showed complete resistance (no susceptibility) to all the six test agents. Among the pathogens tested, Mycobacterium smegmatis was the most resistant, with little susceptibility to the test compounds used. However, Pseudomonas aeruginosa possessed the least resistance, followed by Staphylococcus aureus, then by Escherichia coli, and Klebsiella pneumoniae. Compound 2 showed the most effective antimicrobial activity as it inhibited the growth of Pseudomonas aeruginosa at 0.0125 µg/mL and Staphylococcus aureus at 31.25 µg/mL. It also showed moderate antimicrobial activity against Escherichia coli (250 µg/mL). Compounds 4 and 6 exhibited comparable antimicrobial activity against Pseudomonas aeruginosa with an MIC of 1.95 µg/mL. Staphylococcus aureus strain was more susceptible to compound 3 at MIC 62.5 µg/mL. e compounds did not show any antimycobacterial activity against Mycobacterium smegmatis with the exception of compound 4 at 500 µg/mL. e MICs of the compounds from the HT-SPOTi assay are shown in Table 1.

Resistant Modulatory Studies.
Combination therapy is employed in some microbial infections as a standard option or plans to achieve treatment goals. Resistance modulation investigations offer an advantage in the study of the modulating effect of potential lead compounds on the activities of already available antimicrobials [15]. e nature of the therapeutic effects of potential modulating agents in preventing antimicrobial resistance needs to be investigated through the establishment of the fractional inhibitory concentration index (FICI) to make an intended combination therapy effective in achieving the required treatment outcomes [16].
Antimicrobial resistance modulatory activity study was carried out using the combination assay model for the three most active test compounds P-MEOXY (1), 2ET-TOL (2), and RABYL-TOL (4) against the two most susceptible pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, to investigate their synergistic or antagonistic effects, based on the results obtained from the susceptibility studies.
From the FICI data obtained, compounds 2ET-TOL (2) and RABYL-TOL (4) successfully modulated the activities of amoxicillin against some of the test organisms. It was observed that the combination of compound 4 with amoxicillin exhibited a synergistic effect against Pseudomonas aeruginosa but showed no interaction against Staphylococcus aureus. However, the combination of compound 2 with amoxicillin exhibited an antagonist effect against Staphylococcus aureus and showed no interaction against Pseudomonas aeruginosa. No interaction against Pseudomonas aeruginosa was observed with a combination of compound 1 with amoxicillin ( Table 2).

Biofilm Inhibition Studies.
Microorganisms develop resistance against antimicrobial agents through several mechanisms including the formation of biofilms [17]. Bacteria biofilms constitute about 65% of microbial infections, and those existing in them develop resistance to antimicrobial agents a thousand folds than bacteria existing in planktonic forms [18]. Potential antimicrobial agents should possess strong biofilm inhibition or reduction effects to be considered good candidates in antimicrobial-resistant drug development [19,20]. e test compounds showed variable effects on the inhibition of biofilms in the strains of organisms used at subinhibitory concentrations with P. aeruginosa biofilm being more susceptible to the compounds compared to S. aureus, from the biofilm inhibition studies (Figure 3). e biofilm inhibitory activity against P. aeruginosa was observed to be in a concentration-dependent manner for P-MEOXY (1), P-Z (6), RABYL-TOL (4), and concentration-independent manner for 2ET-TOL (2) indicating different susceptibility patterns of P. aeruginosa biofilms to the compounds. e concentration-dependent effect of RABYL-TOL revealed higher activity with lower concentrations (1/ 16 MIC >1/8 MIC >1/4 MIC >1/2 MIC) (Figure 3(a)). The good activity was observed against P. aeruginosa biofilms with P-MEOXY (at 1/2, 1/4, 1/8 MICs), 2ET-TOL (at all concentrations), and P-Z (at 1/2, 1/4 MICs) (Figure 3(a)). However, all the test compounds exhibited a concentration depending on biofilm inhibition against S. aureus, except P-Z which showed a concentration-independent effect. P-MEOXY showed good biofilm inhibition at 1/2 MIC, and 1/4 MIC against S. aureus biofilm (Figure 3(b)).

Structure-Activity Relationship of the Phosphonium Salts.
Compound 1 (P-MEOXY) which is a hybrid with the methoxy groups at the para position on the triphenyl ring tethered to the triazole and its counterpart compound 2  (1) 1 (NI) -Amoxicillin + 2ET-TOL (2) 0.75 (NI) 6 (AT) Amoxicillin + RABYL-TOL (4) 0 (2ET-TOL) which has methyl groups at the same positions exhibited impressive activity against Gram-negative Pseudomonas aeruginosa followed by it. However, compound 1 performed poorly against the Gram-positive organisms and Mycobacterium tuberculosis. is could be explained by the positive inductive effect of the methoxy and methyl groups which dispersed the positive charge on the phosphonium head making the quaternary group not so essential for activity against Gram-negatives. However, of the hybrids, it was observed that the 2ET-FLU had the least activity generally but performed better than compound 1 against Staphylococcus aureus. is could suggest that the negative inductive effect due to the electron-withdrawing nature of the fluorine group created a more positive center on the phosphonium head which was essential for better activity against Staphylococcus aureus. With the nonhybrids, the compounds 4 (RABYL-TOL) and 6 (PZ) which had substituted phosphonium heads with methyl and methoxy groups, respectively, generally exhibited better activity than PPh3 which possessed an unsubstituted phosphonium head.
is suggests that the electronic effect on the quaternary phosphonium head played a crucial role in influencing the antibacterial activity of the compounds.

Conclusion
A library of six (6) phosphonium salts including three hybrids was synthesized through the SN 2 reaction between the triphenylphosphine derivatives and aromatic benzyl bromides. Click chemistry was however used to synthesize the 1,2,3triazole intermediates. Compound 2 showed the most effective antimicrobial activity as it inhibited the growth of Pseudomonas aeruginosa at 0.0125 µg/mL and Staphylococcus aureus at 31.25 µg/mL. e compounds did not show any antimycobacterial activity against Mycobacterium smegmatis with the exception of compound 4 at 500 µg/mL. From the FICI data, compounds 2ET-TOL (2) and RABYL-TOL (4) successfully modulated the activities of amoxicillin against some of the test organisms. All the test compounds exhibited a concentration-dependent biofilm inhibition against S. aureus, except P-Z (compound 6) which showed a concentrationindependent effect, and good activity was observed against P. aeruginosa biofilms with P-MEOXY (compound 1) (at 1/2, 1/4, 1/8 MICs), 2ET-TOL at all concentrations, and P-Z (compound 6) (at 1/2, 1/4 MICs). e antibacterial activities of the library of the phosphonium salts provide strong proof as scaffolds for future synthetic optimization.