Enantioselective recognition of sutezolid by cyclodextrin modified non-aqueous capillary electrophoresis and explanation of complex formation by means of infrared spectroscopy, NMR and molecular modelling

Highlights

• CD-NACE enantioseparation of sutezolid, linezolid and its precursor.

• Enantiospecific host:guest interactions by spectroscopic approach, FT-IR, NMR.

• Molecular modelling were investigated to provide information about complex formation.

Abstract

A method for the enantioseparation of sutezolid, the next analogue after linezolid and tedizolid, belonging to the truly new class of antibacterial agents, the oxazolidinones, was developed based on non-aqueous capillary electrophoresis (NACE), using a single isomer of cyclodextrins as a chiral pseudophase. During the experiment, the enantioseparation of sutezolid together with its predecessor, linezolid, both weak base antibacterial agents, was evaluated using anionic single-isomers of cyclodextrins from hydrophilic, up to hydrophobic: heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin, heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD), as well as heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD), respectively. Based on the observed results, the cyclodextrins, HDAS-β-CD and HDMS-β-CD which carry the acetyl and methyl groups at the C2 and C3 positions, respectively, provided the baseline separation of sutezolid enantiomers. However, HDMS-β-CD led to a reversal of enantiomer migration order (EMO) in comparison to HDAS-β-CD. Instead, enantiomers of linezolid were separated only by HDMS-β-CD. During the experiments, different organic solvents and their mixtures in various ratios were tested. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the type of organic solvent, and the concentrations of trifluoroacetic acid (TFA) in the NACE buffer system.

Focusing on the desired EMO in which the eutomers (S)-sutezolid and (S)-linezolid migrated last, the highest enantioresolution using the NACE method was achieved at normal polarity mode with 45  mM HDMS-β-CD dissolved in MeOH/ACN (85:15, v/v) containing 200  mM TFA/20  mM ammonium formate. Moreover, infrared spectroscopy, NMR and molecular modelling were investigated to provide information about complex formation.

Introduction

Sutezolid (STD, PNU 100,480), N-({(5S)-3-[3-fluoro-4-(thiomorpholin-4-yl)phenyl]-2-oxo-oxazolidin-5-yl}methyl) acetamide, (Fig. 1A) is the next analogue after linezolid (LIN, Fig. 1B) and tedizolid (TED) and it belongs to a truly new class of antibacterial agents, oxazolidinones [1]. These are, undoubtedly, the most anticipated class of antimicrobial agents, taking into account one of the burning issues in human health, namely, the rapid spread of antibiotic-resistant pathogenic bacteria. Sutezolid is currently in Phase 2 development and most recently completed Phase 2a clinical trials [2] in patients with drug-sensitive pulmonary tuberculosis.

Sutezolid contains a thiomorpholine ring instead of morpholine ring, which constitutes the difference from its predecessor, LIN. That change produced differences in their physicochemical properties which include, the low water solubility of STD (0.2 mg/mL in water), which precludes STD from intravenous administration. Simultaneously, this modification which causes antimicrobial activity, is superior to that of LIN, which means that STD may become a promising candidate to be developed further as the drug of the last chance, mostly for multidrug and extensively drug-resistant tuberculosis (MDR/XDR-TB).

Sutezolid can be considered to be a weak monobasic compound due to the protonation of the nitrogen atom on the thiomorpholine ring. The single isomer (S)-STD possesses one chiral centre at the C5 position of the oxazolidinone ring substituted with an acetamide group, similar to LIN, which is associated with the antibacterial activity of both drugs [1].

Due to the low water solubility of STD, non-aqueous capillary electrophoresis (NACE) became the technique of choice. NACE is a demanding technique; however, it exhibits several interesting features: (i) it offers new possibilities for changes in separation selectivity due to the wide-ranging physical and chemical properties of organic solvents such as the dielectric constant, viscosity, polarity and autoprotolysis; (ii) it produces lower electric currents than aqueous electrolytes, allowing for higher concentrations of the chiral selector; and finally (iii) it facilitates intermolecular interactions among enantiomers and chiral selectors, considering that ion–ion and ion–dipole interactions are stronger at the solvent's low dielectric constant. Thus, small differences between similar analytes become more discriminative in organic solvents than in water [3].

However, there are a few problems with NACE concerns a few items. The first appears at the stage of pH measurement by standard electrodes; in such cases, pH is referred to as “apparent pH” (pH*). The establishment of a pH scale and the determination of the pH in non-aqueous solvents were presented by Porras and Kenndler [4], where the alternative concept of establishing a certain pH was described. Other problems that may arise are related to the significant volatility of some solvents, their dissolving power (which means a solubility limitation for some chiral selectors), UV transparency, and low boiling point.

Due to these factors, methanol and ACN are the most commonly used solvents in NACE. However, single-isomer sulphated CDs are not sufficiently soluble in ACN to permit their use as chiral selectors; hence, the tetrabutylammonium salts of the single-isomer of cyclodextrins (CDs) were synthesised and used for the separation of enantiomers of weak bases in acidic ACN [5].

In NACE, as in the case of aqueous CE, enantioseparation may be achieved by adding different chiral selectors to a NACE buffer system. Among the various chiral selectors, CDs [6] and their derivatives, such as a family of single isomer β- and γ-CD [[7], [8], [9], [10]], are the most widely employed in chiral NACE methods; although macrocyclic antibiotics, proteins, and polysaccharides have also been used [11]. Notably, anionic single isomer CDs at NACE conditions have drawn increasing attention in recent years, as evidenced by numerous publications [[12], [13], [14], [15]]. The resolution mechanism is usually based on inclusion complexation. However, external complex formation with the excellent separation of enantiomers is also possible [12,13]. Methods that can adequately characterise the formation of drug-CD inclusion complexes in solution include not only spectroscopic, electroanalytical and separation techniques, but also polarimetry and isothermal titration calorimetry [16].

This manuscript is part of a project concerning a mechanistic study of the enantioseparation of oxazolidinones. Previous parts of the study have already been published, including the analyses of LIN, TED, and radezolid [[17], [18], [19], [20]]. A better understanding of chirality may contribute to the advancement of our knowledge about non-covalent intermolecular interactions as well as the supramolecular chemistry of the novel analogues of oxazolidinones.

The aim of this study was to develop the principles of chiral separation of STD using charged single isomer derivatives of CDs by NACE and to attempt to explain and understand the process of complex formation. The simultaneous chiral separation of STD and LIN enantiomers was investigated to directly define the influence of the structure of the morpholine ring or thiomorpholine ring (in fact, the difference was one atom) on the complexation process. What is more, enantioselective interactions towards the synthesised precursor of the LIN and STD isomers (preLS) were also studied.

As capillary electrophoresis suffers from the disadvantage of not being able to provide any direct structural information about complex formation, infrared spectroscopy (FT-IR), NMR spectroscopy and molecular modelling were used to understand the complex geometry of (S)-STD and its intermolecular interaction.