Katarzyna Michalska

Recent development of potent analogues of oxazolidinone antibacterial agents

The oxazolidinones are a new and potent class of antimicrobial agents with activity mainly against Gram-positive strains. The commercial success of linezolid, the only FDA-approved oxazolidinone, has prompted many pharmaceutical companies to devote resources to this area of investigation. Until now, four types of chemical modifications of linezolid and oxazolidinone-type antibacterial agents, including modification on each of the A-(oxazolidinone), B-(phenyl), and C-(morpholine) rings as well as the C-5 side chain of the A-ring substructure, have been described. Division into sections according to side chain modification or the type of ring will be used throughout this review, although the process of synthesis usually involves the simultaneous modification of several elements of the linezolid substructure; therefore, assignment into the appropriate section depends on the structure-activity relationships (SAR) studies. This review makes an attempt to summarise the work carried out in the period from 2006 until mid-2012.

NMR and molecular modeling study, as complementary techniques to capillary electrophoresis method to elucidate the separation mechanism of linezolid enantiomers.

NMR study and molecular modeling were performed to improve the level of understanding of the chiral recognition process occurring between linezolid and anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD). NMR spectrometry allowed to estimate the stoichiometry of the complexes between HDAS-beta-CD and S- or R-linezolid and to determine the binding constants. The 1:1 complex stoichiometry was detected in millimolar concentrations and the mode of binding was proposed. The binding constants Ka of the complexes were of the order of 30-80 M(-1). Molecular dynamic simulations of 40ns for four complexes and calculations of binding free energies were performed. These calculations allowed determining the mode of binding of linezolid to HDAS-beta-CD and explaining the binding enantioselectivity.

Determination of linezolid and its achiral impurities using sweeping preconcentration by micellar capillary electrophoresis.

Linezolid is the first compound of a truly new class of antibiotics--the oxazolidinones. The elaborated method of capillary electrophoresis (CE) of linezolid separation from its achiral impurities was successfully performed using sweeping preconcentration, followed by UV absorption detection at 254nm. The best results were obtained with 125mM Tris buffer, pH 2.0, with addition of 20% (v/v) methanol as background electrolyte. Sodium dodecyl sulfate (150mM) was added to the electrolyte in the inlet vial as the sweeping agent. The separation was carried out at negative polarity. Then, the optimized method was validated in terms of linearity, accuracy and precision. Sweeping preconcentration of linezolid provides detection limit at 0.05microg/ml level. The evaluated CE method was applied in the analysis of medicinal product containing linezolid-linezolid solution for infusion.

Determination of enantiomeric impurity of linezolid by capillary electrophoresis using heptakis-(2,3-diacetyl-6-sulfato)-β-cyclodextrin

A method for the enantioseparation of linezolid, the first compound of a truly new class of antibiotics-the oxazolidinones, was developed. The elaborated method of linezolid enantiomers separation was successfully performed using an anionic single-isomer cyclodextrin-heptakis-(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) as a resolving agent with the help of the charged resolving agent migration model (CHARM model). The best results were obtained with 27.5mM HDAS-beta-CD dissolved in 50mM borate buffer, pH 9.0, 15 degrees C, normal polarity. The facile strategies for the reversal of the enantiomers elution order are also described. Afterwards, the optimized method was validated in terms of sensitivity, linearity, accuracy and precision.

Different sample stacking strategies for the determination of ertapenem and its impurities by micellar electrokinetic chromatography in pharmaceutical formulation.

Ertapenem, a Group 1 carbapenem, is most recently introduced into the market. It is a beta-lactam antibiotic that possesses a broad antibacterial spectrum including common community-acquired Gram-positive and Gram-negative aerobic and anaerobic pathogens, but low activity against some nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter spp., enterococci and methicillin-resistant staphylococci. The elaborated method of micellar electrokinetic chromatography (MEKC) of ertapenem separation from its impurities was successfully performed using normal stacking mode (NSM) and stacking with reverse migrating micelles (SRMM), followed by UV absorption detection at 214 nm. The best results were obtained with 60mM sodium dihydrogen phosphate and 20mM boric acid buffer pH 6.0, as background electrolyte. Uncoated fused-silica capillary and neutral-coated capillary with normal and reverse polarity, and voltage values of +18 and -12 kV, respectively, were used throughout the investigation. Sodium dodecyl sulfate was employed as the pseudostationary phase. A comparison of applied techniques, including sensitivity enhancement factors and limits of detection (LOD), is presented. The optimized method was validated in terms of linearity, accuracy and precision. Comparable LOD was obtained using both stacking methods (0.3 microg/mL) but better efficiency of ertapenem peak was obtained using NSM. Under the optimum stacking conditions, about 183-4.75-fold and 1289-4.07-fold improvements in peak areas were obtained for NSM and SRMM, respectively, compared to the usual hydrodynamic sample injection (10s). The reproducibility, expressed by relative standard deviations (RSD) of the migration times, for NSM was about 0.96-1.25 and for SRMM was 0.32-0.45. The RSD of corrected peak areas, for NSM was about 1.07-8.14 and for SRMM was 0.74-8.12. The difference in separation time between the two techniques was not obvious. Satisfactory separation was possible after less than 11min of electrophoresis. The evaluated MEKC method was applied in the analysis of medicinal product containing ertapenem: Invanz-ertapenem for injection.

Determination of doripenem and related substances in medicinal product using capillary electrophoresis

Doripenem, the latest carbapenem antibiotic licensed in the United States (15 October 2007) and the European Union (25 July 2008), has been implemented into therapeutic use along with imipenem, meropenem and ertapenem. The described method of zone electrophoresis in a low pH buffer for the separation of doripenem from its impurities has been successfully performed using field-amplified sample stacking (FASS), followed by UV absorption detection at 214 nm. The best results were obtained with phosphate buffer (100 mM) pH 2.9 containing 10% (v/v) of methanol, as the background electrolyte. Uncoated fused-silica capillary (60/52 cm; 75 μm id) with normal polarity, and voltage values of 25 kV, was used throughout the investigation. The optimised method of doripenem determination was validated in terms of linearity, accuracy and precision, and provides a detection limit of 3.0 μg/mL of doripenem. The repeatability, expressed by relative standard deviation (RSD) of the migration time, for doripenem and its degradation products varied from 1.37 to 2.51%, whereas the corrected peak areas were about 0.91-9.87%. Satisfactory separation was achieved within 20 min of electrophoresis; moreover, all carbapenems (imipenem, meropenem, ertapenem and doripenem) were well separated from each other during this time. The evaluated CZE method was applied in the analysis of a medicinal product containing doripenem Doribax(®) powder for solution for infusion.

Determination of biapenem in a medicinal product by micellar electrokinetic chromatography with sweeping in an enhanced electric field

The elaborated method of micellar electrokinetic chromatography (MEKC) used to separate biapenem from its related substances was successfully implemented using sweeping under an enhanced electric field, followed by UV absorption detection at 200nm. The best results were obtained with formic buffer (22.5mM) pH 4.3 and sodium dodecyl sulfate (150mM) added to the electrolyte as the sweeping agent. Neutral capillary (60/50cm; 50μm ID) with reverse polarity and voltage values of 22kV, were used throughout the investigation. The optimized method of biapenem determination, validated in terms of linearity, accuracy and precision, provides a detection limit of 0.5μg/mL at S/N=3 for biapenem. The repeatability of the CE system, expressed by relative standard deviations (RSD) in the migration times, for biapenem and its degradation products varied from 0.14 to 1.48%, whereas for the corrected peak areas RSD were about 0.68-8.43%. Satisfactory separation was achieved within 20min of electrophoresis; moreover all carbapenems (imipenem, meropenem, ertapenem, doripenem and biapenem) were separated from each other during analysis. The evaluated MEKC method was applied to the analysis of a medicinal product containing biapenem - Omegacin(®) 0.3g for intravenous drip infusion.

Chiral separation of tedizolid using charge single isomer derivatives of cyclodextrins by capillary electrokinetic chromatography.

A method to enantioseparate tedizolid (TED), the second analogue after linezolid (LIN) in a truly new class of antibacterial agents, the oxazolidinones, was developed based on capillary electrokinetic chromatography using cyclodextrin as chiral pseudophase (CD-cEKC). The single isomer R-tedizolid possesses one chiral centre at C5 of the oxazolidinone ring, which is associated with the antibacterial activity of the drug. Tedizolid enantiomers are non-charged and therefore require the use of charged cyclodextrins (CCDs) as carrier hosts to achieve a velocity difference during migration. During method development, hydrophilic anionic single-isomer and moderately hydrophobic and hydrophobic cyclodextrins were tested, including heptakis-(2,3-dihydroxy-6-sulfo)-β-cyclodextrin (HS-β-CD), heptakis-(2,3-diacetyl-6-sulfo)-β-cyclodextrin (HDAS-β-CD), oktakis-(2,3-diacetyl-6-sulfo)-γ-cyclodextrin (ODAS-γ-CD) and heptakis-(2,3-dimethyl-6-sulfo)-β-cyclodextrin (HDMS-β-CD). Only CDs that have acetyl groups at the C2 and C3 positions with seven (HDAS-β-CD) or eight (ODAS-γ-CD) residues of glucopyranose units provided baseline separation of the tedizolid enantiomers with the addition of organic solvent. During the experiments, different organic solvents were tested, such as methanol, acetonitrile, tetrahydrofuran, which varied in their abilities to donate or accept protons. The best enantiomer separation results were obtained using the CD-cEKC method with 37.5mM HDAS-β-CD dissolved in 50mM formic buffer (pH 4.0) with the addition of acetonitrile (81.4:18.6, v/v) at 27ºC, normal polarity, and 12kV. Finally, the apparent binding constants for each enantiomer-HDAS-β-CD pair were calculated. Moreover, in order to evaluate the behaviour of TED and LIN enantiomers relative to chiral selector, enantioselective interactions towards the precursors of TED and LIN isomers were also investigated.

Enantioselective recognition of radezolid by cyclodextrin modified capillary electrokinetic chromatography and electronic circular dichroism

HIGHLIGHTSCEKC enantioseparation of radezolid, linezolid and its precursor.Determination of the apparent average binding constant formation.Enantiospecific host:guest interactions by UV‐CD of the RAD isomers. ABSTRACT A method for the enantioseparation of radezolid (RAD), an analogue of a truly new class of antibacterial agents, oxazolidinones, was developed based on capillary electrokinetic chromatography using a cyclodextrin as a chiral pseudophase (CD‐cEKC). The mechanism of RAD separation, together with its precursor, were investigated to directly define the relationship between the oxazolidinone structure and the complexation process. During the development of the method, anionic single isomer cyclodextrins were tested. They were ranked in order from hydrophilic to hydrophobic as follows: heptakis‐(2,3‐dihydroxy‐6‐sulfo)‐&bgr;‐cyclodextrin (HS‐&bgr;‐CD), heptakis‐(2,3‐diacetyl‐6‐sulfo)‐&bgr;‐cyclodextrin (HDAS‐&bgr;‐CD) and heptakis‐(2,3‐dimethyl‐6‐sulfo)‐&bgr;‐cyclodextrin (HDMS‐&bgr;‐CD). Experiments were performed at pH values of 2.5, 6.6, 8.2 and 9.6. The cyclodextrins that had an acetyl or methyl group at the C2 and C3 positions, referred to as HDAS‐&bgr;‐CD and HDMS‐&bgr;‐CD, respectively, exhibited partial and baseline separation of enantiomers in a low pH buffer. However, higher temperatures were required for HDAS‐&bgr;‐CD and acetonitrile addition was required for HDMS‐&bgr;‐CD. During the experiments, different organic solvents, varying in their amphiprotic or aprotic nature, were tested. The best results for the separation of enantiomers using the CD‐cEKC method were obtained with 40 mM HDMS‐&bgr;‐CD dissolved in a 50 mM phosphate buffer (pH 2.5) with the addition of acetonitrile (65:35, v/v) at 27 °C, reversed polarity and a voltage equal to 28 kV. The apparent binding constants for each enantiomer to HDAS‐&bgr;‐CD or HDMS‐&bgr;‐CD were calculated. Finally, the stereochemistry of (S) and (R)‐RAD and the behaviour of selected complex formations were established using electronic circular dichroism.

Comprehensive spectral identification of key intermediates to the final product of the chiral pool synthesis of radezolid

Radezolid (RAD, 12), biaryl oxazolidinone, was synthesised with small modifications according to the methods described in the literature. The pharmacological activity is observed only for (S)-enantiomer, therefore its synthesis is oriented towards obtaining a single isomer of required purity and desired optical configuration. The intermediate products of RAD synthesis were characterised using 1H- and 13C-NMR, as well as the 2D correlation HSQC and HMBC (2, 5, 9, 10), furthermore studied using infrared radiation (FT-IR), Raman scattering (3, 5, 9), and electronic circular dichroism (ECD) (5, 12) spectroscopy. Each technique provides a unique and specific set of information. Hence, the full spectral characteristics of key intermediates obtained from the chiral pool synthesis to the finished product of RAD were summarised and compared. For a more accurate analysis, and due to the lack of reliable and reproducible reference standards for intermediate products, their vibrational analysis was supported by quantum chemical calculations based on the density functional theory (DFT) utilising the B3LYP hybrid functional and the 6-311G(d,p) basis set. Good agreement was observed between the empirical and theoretical spectra.Graphical abstractComprehensive spectral identification (ECD, NMR, FT-IR, Raman) of key intermediates of the chiral pool synthesis of radezolid.