A Study of Fluorinated Β-nitrostyrenes as Antimicrobial Agents

The effect of variously fluorine-substituted β-methyl-β-nitrostyrenes on their antimicrobial activity was investigated. Their efficacy was determined by minimum inhibition concentration (MIC) in cultures of Gram positive and Gram negative bacteria and a fungus. Highest activity against the Gram negative bacterium, E.coli, was achieved with 4-fluorine-aryl substituted β-methyl-β-nitrostyrenes, while most compounds gave excellent results against gram positive bacteria. Importantly, the addition of the β-methyl group profoundly enhanced the antibacterial activity of the compounds tested. The comparative K D values for the most potent compounds against E.coli were much lower than those required for the gram positive and fungus counterparts. This investigation illustrated that fluorine substituted nitropropenylarenes have enhanced antimicrobial activity suitable for antibiotic applications.


Introduction
Time is running out for antibiotics.The rising levels of antibacterial resistance, the decline in productive drug development and the reduction in the numbers of pharmaceutical companies engaging in research and development of anti-infective agents are huge public health concerns and pose problems for the effective management of microbial infections [1].There is an urgent need to source OPEN ACCESS novel antimicrobial agents to combat human pathogens that are not susceptible to currently available antibiotics coupled with the strategic use of all available and emerging antimicrobial agents.
In 1952 Schales and Graefe [2] found that a range of substituted β-nitrostyrenes including nitropropenyl aromatic compounds were active against Gram positive and Gram negative bacteria and Denisenko et al. [3] who confirmed that 1 was as active as clinical antibiotics against bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).From the work of Milhazes et al. in 2006, the antibacterial activity against S aureus American Type Culture Collection (ATCC) 29213 of some aryl-hydroxy/methoxy substituted β-nitrostyrenes was found to be improved by the addition of a β-methyl group on the nitro alkene, with a 2 to 8 fold increase in the minimum inhibitory concentrations (MIC) relative to the β-nitrostyrene [4].Interestingly, this MIC enhancement was most pronounced for Gram positive bacteria utilizing 3-hydroxy-4-methoxy-β-methyl-β-nitrostyrene 2 (MIC 32); whereas 3,4-dihydroxy-β-methyl-βnitrostyrene 3 (MIC 64) showed the best result against all Gram negative bacteria except for Pseudomonas aeruginosa.
Protein-tyrosine phosphatases (PTPases) are modulators of signal transduction pathways that regulate numerous cell functions.Malfunction of PTPases has been linked to a number of oncogenic and metabolic disease states.The PTPases are also utilized by microbes and viruses for pathogenic activity.Therefore, inhibitors of PTPases have potential as therapeutic antimicrobial agents [5][6][7].Park and Pei in enzyme mechanistic studies found trans-β-nitrostyrene derivatives to be a new class of inhibitors of protein tyrosine phosphatase PTP1B [8].They proposed that β-nitrostyrene is a reversible inhibitor of the tyrosine phosphatases PTP1B by their interaction and formation of a covalent complex with cysteine at the catalytic site.Scheme 1. Park and Pei [8] mechanism of PTP1B inhibition by β-nitrostyrene.Reprinted with permission from Park, J.; Pei, D. Trans-β-nitrostyrene Derivatives as Slow-Binding Inhibitors of Protein Tyrosine Phosphatases.Biochem.2004, 43, 15014-15021.Copyright 2004, American Chemical Society.

S
Their evidence suggested that in the absence of free thiols, nucleophilic attack by enzymic cysteine occurs on the side chain nitro group, as shown in Scheme 1.This rationale was based on the positive charge on the nitrogen atom being susceptible to nucleophilic attack, forming a reversible adduct that inhibited PTP1B.However, exactly the opposite is found in the literature.It has been reported [9] that β-nitrostyrenes reversibly add to the thiol group of cysteine and cysteine peptides under mild conditions.In contrast, sodium trimethylsilane thiolate at 185 °C for 24 h has been found to reduce nitro groups to amines [10].The conjugate addition to β-nitroalkenes reflects the high reactivity of β-nitroalkenes as Michael acceptors [11], and it is widely recognized that nitro group olefins undergo rapid conjugate addition with thiol-type nucleophiles [12,13].
The antimicrobial activity of a range of β-methyl-β-nitrostyrene derivatives 4 against Gram positive, Gram negative bacteria and fungi were evaluated by Nicoletti et al. [14] who had previously found that: (a) E.coli was suppressed effectively by chlorine or fluorine C 4 -aryl-substituents relative to the unsubstituted or the 3-4-methylenedioxy ring compounds.(b) S.aureus was suppressed effectively by a wide range of nitropropenyl arenes including β-methyl-β-nitrostyrene, the 4-fluorine and 4-chlorine substituted β-methyl-β-nitrostyrene, imidazolyl, benzothiazole and the 3,4-methylenedioxy derivatives.An important finding was that when two hydroxy groups were present on the ring in the 2-and 4-positions or the 2-or 5-positions, activity against this microorganism noticeably reduced.The fact that this also occurred with substitution by N,N-dimethyl and N,N-diethyl groups indicated that the more polar nature of these substituents was detrimental to activity.This was supported by the K D values of the latter compounds being relatively low compared with the unsubstituted and halogenated-substituted compounds.(c) B.subtilis was suppressed by a wide range of compounds in a similar fashion to S.aureus.The 2,4-and 2,5-dihydroxyaryl substituted compounds exhibited poor bacterial inhibition whereas the 3,4-dihydroxy derivative gave high activity.(d) C.albicans was suppressed by the 3,4-dichloro, 4-chloro, 4-fluoro, derivatives.
The introduction of 'fluorine functionality' into molecular architecture has proved to be invaluable for modulation of the molecular properties of medicinal compounds [15].The substitution of hydrogen and other functional groups with fluorine can have multiple molecular impacts, including electronic, lipophilic, H-bonding, chemical, metabolic stability and steric factors, which can influence the pharmacodynamic and pharmacokinetic properties of pharmaceuticals [16].When fluorine atoms or trifluoromethyl groups were introduced into either the peptidic chain or the C-terminal end of cationic pentapeptides, the antimicrobial activity always improved by diminishing the minimal inhibitory concentration (MIC) [17].A recent report on the structure activity relationships of antitubercular nitroimidazoles highlighted the high activity achieved by the inclusion of trifluoromethoxybenzyl substituents in the nitroimidazoles [18].Carbon films with a high fluorine content exhibited excellent antibacterial properties against E. coli growth [19].Furthermore, a simple super hydrophobic coating composed of nano-structured fluorinated silica colloids with fluoroalkoxysilane on a silane structure demonstrated that the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa was reduced by 2.08 ± 0.25 and 1.76 ± 0.12 log over controls, respectively [20].The fluorine containing 4-(substituted-2-hydroxybenzoyl) pyrazoles 6, 7, 8 showed promising antibacterial activity [21].Based on the discoveries that fluorine substituted β-nitrostyrene enhanced antimicrobial activity, this investigation was undertaken to study the effects on antimicrobial activity of difluoro-, trifluoromethyl-and trifluoromethoxy-substituted β-methyl-β-nitrostyrenes and related compounds that were prepared via the Henry reaction [22].For an antimicrobial agent to be effective, it must penetrate the bacterial cell first to reach its target enzymes, therefore the lipophilicity of the prepared compounds was determined from the octanol-water partition coefficients.The antimicrobial activities of all products were measured by minimum inhibitory concentration (MIC).Three Gram positive bacteria (Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis), one Gram negative bacterium (Escherichia coli) and a fungus (Candida albicans) were tested.

Chemicals
Chemicals and solvents were typically sourced from Sigma-Aldrich Chemical Company, and were AR grade or better.
NMR spectroscopy was performed on Bruker Advance 300 MHz or Bruker Avance 300 III MHz spectrometer.Some FIDs from Bruker Advance 300 MHz were processed using Mestrec23. 1H chemical shifts were recorded as δ values in parts per million (ppm) downfield shifts, referenced internally to the residual CHCl 3 singlet at 7.26 ppm.Chemical shifts are presented in multiplicity, coupling constant(s) (J in Hz), integration and assignments.Abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, dd = doublet of doublet, m = multiplet. 13C chemical shifts were 1 H decoupled and recorded as δ values in parts per million (ppm), referenced internally to the CHCl 3 triplet at 77.0 ppm.Assignments have typically been made using additional information from COSY and gCOSY spectra, DEPT, HMBC and HSQC experiments.
Melting Points were determined on a Gallenkamp melting point apparatus, and were uncorrected.

Minimum Inhibitory Concentrations
The strains used for biological tests were: Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231.Stocks of all microorganisms were kept in −80 °C in MHB (Oxoid, Cambridge, UK) with 20% v/v glycerol (BDH chemicals, Poole, UK).Strains were subcultured onto Nutrient agar (NA, Oxoid) and subcultures between three and nine used to prepare inocula.Inocula were prepared by adjusting a suspension to match McF 0.5 turbidity standard (equal to ~1.5 × 10 8 bacterial cells/mL or ~1.5 × 10 6 yeast cells/mL) and subsequent dilution to the required density.Broth microdilution testing was according to the National Committee for Clinical Laboratory Standards standard methods [26,27] in MHB (Oxoid) for bacteria or Sabouraud Liquid Medium (SLM, Oxoid) for C. albicans.Microplate assays were performed in clear, round-bottomed, 96-well plates (Sarstedt Australia, SA, AUS) with a total volume of 200 µL per well.Standard inoculum densities were approximately 1 × 10 5 Colony Forming Units per mL (CFU/mL) for bacteria and 1 × 10 4 CFU/mL for C. albicans.Inoculum densities were confirmed by serial dilution plating onto NA and incubation aerobically at 37 C for 24 h.
The test β-nitrostyrene derivatives were added to plates at two times tested concentrations in 100 μL media.Ciprofloxacin was used as an internal positive control for bacteria and miconazole for C.albicans.Microplates were incubated 18-24 h at 37 °C aerobically before reading wells visually for turbidity.All assays included duplicated wells and were at least twice replicated.The geometric MIC (μg/mL) for each strain was adjusted to the nearest log 2 dilution tested.MIC results were reported as MIC (μg/mL) for standards.

Octanol-Water Partition Coefficients
The lipophilicity level of each compound was determined by octanol-water partition coefficients.The buffer solution used in the determination of K D was made by mixing sodium chloride (3.78 g, 65 mmol), disodium hydrogen orthophosphate (2.14 g, 18 mmol) and sodium dihydrogen orthophosphate (0.78 g, 5.5 mmol) in 500 mL water at room temperature (~22 °C) and had a pH of 7.5.Each compound (10 mg) was dissolved in octanol (2 mL) in a stoppered test tube, followed with the addition of the buffer solution (2 mL) and the tube was shaken over 48 hours.Finally the mixtures were allowed to separate into two layers.The top layers were removed and absorbance measured after dilution 1:20, 1:50 or 1:200 to 3 mL with octanol in a 1 cm cuvette path length at 370 nm for each diluted sample.The aqueous bottom layers were removed and the absorbance measured without dilution.K D measurements were according to the equation:

K Octanol Water
As the absorbance of the octanol and water layers is directly proportional to the concentration in each layer, the K D value can be calculated from the relative absorbance of each layer.

Results and Discussion
The preparation of the β-methyl-β-nitrostyrenes involved the Henry condensation reaction [22] of the aromatic aldehyde with nitroethane under basic conditions followed by dehydration of the resultant alcohol.The NMR analysis of the β-methyl-β-nitrostyrene fluorine derivatives indicated that the β-methyl-β-E-nitrostyrene conformation was predominant in most of the compounds prepared.For example, for 13 the E/Z ratio is 14/1.
The antibacterial activity of β-nitrostyrene derivatives and related compounds has been well known for a number of years.Their resurgence is attributed to the strong antibacterial/antifungal activity of some compounds with applications in veterinary and human medicine.It has been established from SAR studies that their lipohilicity plus the nucleophilic addition of thiol groups of some enzymes to the exocyclic double bond of these compounds are critical for the development of their antimicrobial activity.The antimicrobial efficacy of the β-nitrostyrenes was assessed by their activity against a panel of bacteria and a fungus (Candida albicans).The results appear as the minimum inhibitory concentration (MIC) for each compound.The partition coefficient (K D ) between octanol and water for each compound, representing its degree of lipophilicity, was also determined in order to access the extent of its interaction with the surface of the microorganism.The measured and calculated log P values are also included.Compounds with the incorporation of fluorine were of considerable interest as earlier studies had indicated an improvement of antibacterial activity [9].
The antibacterial assays shown in Table 1 indicated that the β-nitrostyrene compounds 20 and 9 displayed significantly lower activity towards all the tested bacteria relative to all the β-methyl-β-nitrostyrene analogues that were studied.This is exemplified by comparing the activity values of 20 and 21 as well as 9 and 5 that differ by at least an order of magnitude.The β-nitropropenyl side chain deviates 28° from coplanarity with the benzene ring [4] and the relative higher K D values of the β-methyl-β-nitrostyrene compounds provides evidence that the steric effect and presence of the β-methyl group is substantial.However, the reasons for their enhanced antibacterial potency are unclear.It is also difficult to correlate the position of fluorine substitution on the benzene ring or the effect of multiple fluorine substituents with activities and K D values of 5, 10 11 and 12.The activity order of the four most potent antibacterial compounds 5 > 10 > 12 > 13 does not correlate with their K D values.The data also provides evidence of the apparent tendency for fluorine functionalized β-methyl-β-nitrostyrenes to show considerable hydrophilic properties.The results also suggest that the more hydrophilic nature of the fluorine functionalized-β-methyl-β-nitrostyrenes with K D values in the lowest range ) is more effective against E.coli.It is interesting to note that substituent size appears to affect antibacterial activity against E. coli., since for F-, the potency order is p > o > m and for CF 3 -, its p > m > o.Of all the substances tested, compound 5 was most active against E.coli (27 µg/mL) and the MIC values against the other microorganisms were 6 µg/mL or less, making it also the most effective against the three Gram positive bacteria and C.albicans.We deduce from this that for many Gram negative bacteria (such as E. coli) that are known to have polysaccharide structures, hydrophilic compounds more readily penetrate the bacterial cell wall to inhibit bacterial enzymes.From our results, the only Log P value for that can be cited for an effective fluorinated compound on E.coli (Gram negative) is 2.00 for compound 5.For the Gram positive bacteria, a range of Log P values of 1.15-2.19apparently relate to efficacy.For the non-fluorinated compounds, optimal Log P values cover a wider range 1.61-3.41,and becomes wider 1.15-3.41with the inclusion of C.albicans.The plots of the MIC-K D for the twelve β-methyl-β-nitrostyrenes investigated for their antibacterial activities against E. coli and E. faecalis presented in Figure 1 reflect the poor correlation between the effectiveness against E.coli and the partition coefficients of the compounds tested (r 2 = 0.0498 for E.coli, r 2 = 0.1416 for E. faecalis).There were no correlations between MIC value and K D value for S. aureus, B. subtilis, and C. albicans (graphs not shown).Can aromatic ring fluorine/hydrogen exchange influence membrane permeability?Generally, aromatic F/H substitution tends to increase compound lipophilicity, in direct contrast to what is observed.Alternatively, the reason for the effectiveness of fluorine substitution on the aromatic ring may be connected with the high electronegativity of fluorine, although size factors could also be important.Perhaps the electronegativity of fluorine could affect binding affinity to the binding site of the bacteria, thus causing inhibition of the enzyme.Further investigations with other fluorine-substituted compounds to determine the structural features required for the optimal anti-bacterial activity of β-methyl-β-nitrostyrene compounds are currently being carried out.

Conclusions
We have deployed the β-methyl-β-nitrostyrene scaffold for the development of fluorinated derivatives with enhanced antibacterial activity.While the screening of whole cells in MIC assays ensures cell penetration and antimicrobial activity, it does not distinguish selective inhibitors from toxic compounds.In addition, the mechanism of antimicrobial action is unknown.The dependency between antimicrobial activity and lipophilicity K D of fluorinated β-methyl-β-nitrostyrenes (FBNS) was examined for three Gram positive and one Gram negative bacteria and a fungus.The antimicrobial activity is rationalized with unspecific cytoplasmic membrane damaging effects that is near optimum at a lipophilicity (logP) of 2 (±0.5) for Gram-negative bacteria.For Gram-positive bacteria and fungus poor MIC vs. log K D correlations were found.The disparity between measured and calculated log P values is also under investigation.There is, however, unequivocal literature evidence that both the β-nitrostyrenes and the β-methyl-β-nitrostyrenes are exceptional Michael acceptors.An expanded set of substituted β-methyl-β-nitrostyrene compounds will be investigated to determine the structural features required for the optimal anti-bacterial activity of β-methyl-β-nitrostyrene compounds.

Table 1 .
Antibacterial/fungus assays of various β-E-nitrostyrene compounds, their MIC [μg/mL] and K D , logP values.MIC and K D values are the means of two determinations.