Glycosides of Nadifloxacin—Synthesis and Antibacterial Activities against Methicillin-Resistant Staphylococcus aureus

The increase in the number of bacteria that are resistant to multiple antibiotics poses a serious clinical problem that threatens the health of humans worldwide. Nadifloxacin (1) is a highly potent antibacterial agent with broad-spectrum activity. However, its poor aqueous solubility has limited its use to topical applications. To increase its solubility, it was glycosylated herein to form a range of trans-linked (3a-e) and cis-linked (7a,b) glycosides, each of which was prepared and purified to afford single anomers. The seven glycoside derivatives (3a-e, 7a,b) were examined for potency against eight strains of S. aureus, four of which were methicillin-resistant. Although less potent than free nadifloxacin (1), the α-L-arabinofuransoside (3a) was effective against all strains that were tested (minimum inhibitory concentrations of 1–8 μg/mL compared to 0.1–0.25 μg/mL for nadifloxacin), demonstrating the potential of this glycoside as an antibacterial agent. Estimation of Log P as well as observations made during preparation of these compounds reveal that the solubilities of the glycosides were greatly improved compared with nadifloxacin (1), raising the prospect of its use in oral applications.


Introduction
Staphylococci are commonly found in the environment and are major colonizers of human skin [1,2]. Staphylococus aureus, the major human pathogen of the genus, is mostly harmless, but in certain situations it can cause severe illness, such as endocarditis, pneumonia, sepsis and toxic shock syndrome [3,4]. Infections were initially treated with benzylpenicillin, but by the late 1950s, resistant strains of the bacteria that produced βlactamase were increasingly being isolated. In 1959, the β-lactamase-resistant antibiotic methicillin was introduced, but since then there has been a steady increase in the prevalence of methicillin-resistant strains of bacteria, and last-resort drugs such as vancomycin are increasingly used to treat infections [5,6]. The isolation of strains with reduced susceptibility to this drug, termed vancomycin-intermediate-resistant S. aureus (VISA), has raised the prospect of there being no antibacterial therapy for such strains [7].
Nadifloxacin (1) (Scheme 1) is a broad-spectrum fluoroquinolone antibiotic that demonstrates high potency against aerobic Gram-positive and Gram-negative organisms and anaerobes. It has also been shown to be highly effective against methicillin-resistant Staphylococcus aureus, and low incidence of resistance is noted [8,9]. Unfortunately, nadifloxacin is poorly soluble in water and can only be used medicinally in topical ointments to treat acne and other skin infections [10,11]. Many attempts have been made to increase the aqueous solubility of nadifloxacin and thus enable it to be used in oral administration, for example by forming carboxylate salts, esters and peptides and by incorporating the drug in microemulsions and dendrimers [12][13][14][15][16][17]. However, the production of a suitable commercial derivative has so far been unsuccessful.
An alternative approach to improve the solubility of nadifloxacin is to glycosylate the free hydroxyl group on the piperidine ring (Scheme 1). Since this hydroxyl group is required
The cis-galactoside (7a) and cis-glucoside (7b) were obtained by changing the reaction solvent and glycoside-protecting groups to favour the formation of the thermodynamic product (Scheme 2). The tetra-O-benzyl glycosides (4a) and (4b) were acetylated with acetic anhydride, then coupled with nadifloxacin (1) using TMSOTf in anhydrous acetone. This afforded a mixture of glycoside anomers with α:β ratios of 1:1.59 for the galactoside (6a) and 1:1.23 for the glucoside (6b), as evidenced by 1 H Nuclear Magnetic Resonance (NMR) spectroscopic analysis. The benzyl ethers were then removed by hydrogenolysis over palladium on charcoal to afford (7a) and (7b) as a mixture of anomers. These anomeric mixtures could not be separated by chromatography, and instead the β-glycoside was selectively hydrolysed using the corresponding β-glycosidase enzymes-β-galactosidase from Escherichia coli in phosphate-buffered saline pH 7.0 or β-glucosidase from almonds, in acetate buffer, pH 6.0. The mixtures were then purified using flash chromatography as described above. All intermediates (2a-e), (5a-b), (6a-b) and deprotected glycosides (3a-e) and (7a,b) were characterised by 1 H and 13 C NMR and IR, spectroscopic analysis and mass spectrometric analysis. Purity was determined by High Performance Liquid Chromatography (HPLC) to be > 97% prior to subsequent analysis of the inhibitory properties of the glycosides (3a-e) and (7a,b). raphy as described above. All intermediates (2a-e), (5a-b), (6a-b) and deprotected glycosides (3a-e) and (7a,b) were characterised by 1 H and 13 C NMR and IR, spectroscopic analysis and mass spectrometric analysis. Purity was determined by High Performance Liquid Chromatography (HPLC) to be > 97% prior to subsequent analysis of the inhibitory properties of the glycosides (3a-e) and (7a,b). Scheme 2. Synthesis and resolution of nadifloxacin-α-galactoside (7a) and α-glucoside (7b).
All of the synthesised nadifloxacin glycosides (3a-e) and (7a,b) were soluble in water at physiological pH as demonstrated in the final enzymatic resolution step in Scheme 2, wherein the product was dissolved in aqueous systems to allow for enzymatic hydrolysis. The octanol-water coefficients (Log P) were also calculated as 0.05 for the hexose glycosides and 0.51 for the pentoses, compared to 1.79 for underivatised nadifloxacin (1) (calculated using ChemDraw 12.0, Perkin-Elmer, Cambridge, UK).
It should be noted that racemic nadifloxacin was used in all glycoside syntheses, and the products (3a-e) and (7a,b) were isolated as mixtures of diastereomers. This afforded more complex NMR spectra than expected with some peaks unresolved and thus quoted as multiplets. Scheme 2. Synthesis and resolution of nadifloxacin-α-galactoside (7a) and α-glucoside (7b).
All of the synthesised nadifloxacin glycosides (3a-e) and (7a,b) were soluble in water at physiological pH as demonstrated in the final enzymatic resolution step in Scheme 2, wherein the product was dissolved in aqueous systems to allow for enzymatic hydrolysis. The octanol-water coefficients (Log P) were also calculated as 0.05 for the hexose glycosides and 0.51 for the pentoses, compared to 1.79 for underivatised nadifloxacin (1) (calculated using ChemDraw 12.0, Perkin-Elmer, Cambridge, UK).
It should be noted that racemic nadifloxacin was used in all glycoside syntheses, and the products (3a-e) and (7a,b) were isolated as mixtures of diastereomers. This afforded more complex NMR spectra than expected with some peaks unresolved and thus quoted as multiplets.
Since nadifloxacin (1) was developed as a potent antimicrobial for the treatment of MRSA, the antimicrobial activities of the synthesised nadifloxacin glycosides were determined for a range of Staphylococcus aureas strains. The lowest concentration at which bacterial growth could not be detected was recorded as the minimum inhibitory concentration (MIC). All of the Staphylococcus aureas strains were found to be sensitive to nadifloxacin, and all of the strains were found to be sensitive to, or intermediately sensitive to, the glycosides. However, all of the glycosides (3a-e) and (7a,b) had MICs that were higher than free nadifloxacin (1) (Table 1), presumably due to the requirement of hydrolysis to release the free nadifloxacin. This could indicate that the nadifloxacin glycosides do not induce the genes that produce the proteins required for transport and hydrolysis of the glycosides. There was variation in the MICs between glycosides and between strains for the same glycoside, probably due to differences in the rate of uptake of the glycoside by the bacteria and the amount of glycosidase that was expressed by the bacteria. Free nadifloxacin (1) was slightly more inhibitory for the MRSA strains than for non-MRSA organisms (0.125 µg/mL compared to 0.25-0.5 µg/mL), but there was no general pattern to the resistance of the glycosides. Of the glycosides, the α-L-arabinofuranoside (3a) was the most potent with MICs of 1-8 µg/mL for all of the strains that were examined.

Materials and Methods
NMR spectra were recorded on a Bruker DPX spectrometer (400 MHz), and chemical shifts are quoted in ppm relative to tetramethylsilane as internal standard using the following abbreviations: s, singlet, d, doublet, at, apparent triplet, as, apparent singlet and m, multiplet. Liquid Chromatography Mass Spectrometry (LCMS) was accomplished using a ThermoFisher Scientific Accela LC system coupled to a ThermoFisher Scientific LTQ Fleet Ion Trap Mass Spectrometer (ThermoFisher Scientific, Loughborough, UK). Melting points were recorded on a TA Instruments DSC Q2000 instrument heating at 10 • C/min (Hertfordshire, UK). FTIR spectra were recorded on a ThermoFisher Scientific Nicolet iS10 instrument. Thin-layer chromatography was performed using ALUGRAM SIL G precoated plates (Macherey-Nagel, Germany). The purity of deprotected samples was achieved using an Agilent 1100 series HPLC with a ThermoFisher Scientific Hypersil Gold Column (50 × 4.6 mm, 3 µm) eluting 0.1% (v/v) formic acid in water/MeCN (85:15), 2 mL/min, UV absorbance at 254 nm.

Deprotection of per-O-acetylated nadifloxacin glycosides-General method
The isolated glycosides (2a-e) were de-O-acetylated using a mixture of anhydrous dichloromethane and methanol (1:6) and potassium carbonate (50% w/w) over 30-60 min. The mixtures were adjusted to approximately pH 3 by the addition of formic acid and were purified on C18 columns and eluted with 0.1% (v/v) formic acid in water (A) and acetonitrile (B) isocratically at 7:3.

Deprotection of per-O-acetylated nadifloxacin glycosides-General method
The isolated glycosides (2a-e) were de-O-acetylated using a mixture of anhydrous dichloromethane and methanol (1:6) and potassium carbonate (50% w/w) over 30-60 min. The mixtures were adjusted to approximately pH 3 by the addition of formic acid and were purified on C18 columns and eluted with 0.1% (v/v) formic acid in water (A) and acetonitrile (B) isocratically at 7:3.