Synthesis of a naphthyridone p38 MAP kinase inhibitor

Compound 1 is a p38 MAP kinase inhibitor potentially useful for the treatment of rheumatoid arthritis and psoriasis. A novel six-step synthesis suitable for large-scale preparation was developed in support of a drug development program at Merck Research Laboratories. The key steps include a tandem Heck-lactamization, N-oxidation, and a highly chemoselective Grignard addition of 4-(N-tert-butylpiperidinyl)magnesium chloride to a naphthyridone N-oxide. The N-oxide exerted complete chemoselectivity via chelation in directing the Grignard addition to the alpha position as opposed to 1,4-addition on the ene-lactam. The dihydropyridyl adduct was in situ aromatized with isobutylchloroformate followed by heating in pyridine. Syntheses of Grignard precursor, N-tert-butyl-4-chloro-piperidine, were accomplished via transamination with a quaternary ammonium piperidone or via addition of methylmagnesium chloride to an iminium ion. Utilizing this chemistry, multi-kilogram preparation of compound 1 was successfully demonstrated.     

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).     

Chemometric method to optimize chiral separation of imidazole derivatives by capillary electrophoresis

A chemometric methodology was proposed to optimize the migration time, height equivalent to a theoretical plate and separation of a mixture of a series of imidazole compounds by capillary electrophoresis. The optimization process was based on a special polynomial from 9 or 18 preliminary experiments. This method connects a general simplex method to a computer. A simplex two or three optimization-capillary electrophoresis (STO-CE) method has been developed in our laboratory. The most efficient separation was achieved with acetonitrile-phosphate buffer, pH 4.70, (5.30+94.70 (v/v)) with a beta-cyclodextrin concentration in the background electrolyte equal to 5.80 mM and a capillary temperature of 35 degrees C. Similar results were obtained using simple step-wise scanning. The higher relative difference obtained for these values with these two methods (simplex and step-wise scanning) was 5% for the beta-cyclodextrin concentration factor.     

Development and validation of a chiral capillary electrophoresis method for assay and enantiomeric purity control of pramipexole

A rapid method for the enantioseparation of pramipexole and its R‐enantiomer has been developed by capillary electrophoresis. The influence of chemical and instrumental parameters was investigated including the type and concentration of chiral selectors, buffer composition and pH, co‐ions, applied voltage, capillary length and temperature. Optimal separation conditions were obtained using a 50 mM phosphate buffer (pH 2.8) containing 25 mM carboxymethyl‐β‐cyclodextrin on a fused‐silica capillary. Online UV detection was performed at 262 nm. A voltage of 25 kV was applied, and the capillary temperature was kept at 25°C. Hydrodynamic injection was performed at 3.45 kPa for 5.0 s. The separation of enantiomers was achieved in <6.5 min. The method was further validated in terms of stability of solutions, selectivity, linearity (both pramipexole and R‐enantiomer, R²>0.995), LOD and LOQ (0.91 and 2.94 μg/mL, respectively), repeatability (RSD<1.5%) and accuracy (pramipexole, 100.4%; R‐enantiomer, 100.5%). The proposed method was then applied to two kinds of pramipexole dihydrochloride monohydrate commercially available tablets, immediate release tablets (1.50 and 0.125 mg) and sustained release tablets (0.52 mg), to quantify the main component in the tablets. The amount of distomer could be quantified in bulk sample materials.     

Enantiomeric resolution of chiral imidazole derivatives using capillary electrophoresis with cyclodextrin-type buffer modifiers

Enantiomeric resolutions of some chiral pharmaceuticals containing the imidazole (1,3-diazole) moiety were carried out using capillary electrophoresis. Various native cyclodextrins (ga-, β- and γ-cyclodextrin) and derivatized cyclodextrins (hydroxypropyl-, and sulfobutyl ether-β-cyclodextrin) were used as chiral buffer modifiers. The effects of the cavity size, the structure and the charge of the selectors on the chiral recognition ability were evaluated. The influence of the type and concentration of the organic modifier on the separation of miconazole enantiomers and the pH of the run buffer on the separation of enilconazole enantiomers was also studied.     

Quality by design-guided development of a capillary electrophoresis method for the simultaneous chiral purity determination and impurity profiling of tamsulosin

Analytical Quality by Design principles using the design of experiments were applied for the development of a capillary electrophoresis method for the determination of enantiomeric purity and chemically related impurities of tamsulosin. From initial scouting experiments, a dual cyclodextrin (CD) system composed of sulfated β-CD and carboxymethyl-α-CD was selected as the chiral selector. A fractional factorial resolution V+ design was used for the identification of the critical process parameters, while a face-centered central composite design and Monte Carlo simulations were employed for final optimization and defining the design space of the method. The experimental conditions of the working point were: 30 mM sodium phosphate buffer, pH 3.0, containing 40 mg/mL sulfated β-CD and 7 mg/mL carboxymethyl-α-CD, capillary temperature 18°C, applied voltage -23 kV. Following the assessment of robustness by applying a Plackett-Burman design, the method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use guideline Q2(R1). The method allowed the quantification of the chiral impurity and three other related impurities at the 0.1 % level with acceptable accuracy and precision.     

Development and validation of a robust capillary electrophoresis method for impurity profiling of etomidate including the determination of chiral purity using a dual cyclodextrin system

An enantioselective CE assay for the simultaneous determination of the enantiomeric purity and of related substances of etomidate has been developed and validated using a binary chiral selector system employing 30 mg/mL beta-CD and 4.6 mg/mL sulfated-beta-CD in a 150 mM potassium phosphate buffer, pH 2.1. The method was validated with respect to specificity, range, linearity, LOQ and LOD, precision and accuracy. The assay allowed the detection and determination of related substances including (S)-etomidate at the 0.05% w/w level, the reporting threshold as defined by the International Conference on Harmonisation guidelines as well as the European Pharmacopoeia. Robustness testing was carried out by an "Augmented Plackett-Burman" design. Quantitation of the compounds was performed by calibration graphs with respect to lidocaine hydrochloride as internal standard and by peak area normalization, the procedure usually applied by pharmacopoeias. Although data obtained from the calibration graphs constructed with the aid of the internal standard were more accurate based on compound recovery, peak area normalization may also be used without significant loss of accuracy and precision.     

Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation

Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.     

Synthesis of p38 MAP kinase inhibitor BIRB 796 and analogs via copper-mediated N-arylation reaction

Direct N-arylation of urea (5) with various arylboronic acids mediated by cupric acetate furnished BIRB796 and a range of N-substituted BIRB796 analogs in good to moderate yields in one step. Urea (5) was readily synthesized from commercially available compounds.     

Stability evaluation of tramadol enantiomers using a chiral stability-indicating capillary electrophoresis method and its application to pharmaceutical analysis

In this study, a chiral stability-indicating CE assay was developed for the stability evaluation of tramadol (TR) enantiomers in commercial tablets using maltodextrin as chiral selector. To investigate the stability-indicating power of the analytical method as well as stability evaluation of TR enantiomers, active pharmaceutical ingredient and TR tablets were subjected to photolysis, heat, oxidation and hydrolysis to conduct stress testing. Best separation for the TR enantiomers was achieved on an uncoated fused-silica capillary at 20 °C using borate buffer (50 mM, pH 10.2) containing 10% m/v maltodextrin. All determinations were performed by a UV detector at 214 nm. A constant voltage of 20 kV was applied to obtain the separation. The range of quantitation for both enantiomers was 5-100 μg/mL (R>0.996). Intra- and inter-day RSD (n=6) were less than 10%. The percent relevant errors were obtained to be less than 4.0 for both enantiomers. The limits of quantitation and detection for both enantiomers were 5 and 1.5 μg/mL, respectively. Degradation products resulting from the stress studies were the same for both enantiomers and did not interfere with the detection of the enantiomers.     

Development of a capillary electrophoresis method for the determination of the chiral purity of dextromethorphan by a dual selector system using quality by design methodology

Dextromethorphan is a centrally acting antitussive drug, while its enantiomer levomethorphan is an illicit drug with opioid analgesic effects. As capillary electrophoresis has been proven as an ideal technique for enantiomer analysis, the present study was conducted in order to develop a capillary electrophoresis‐based limit test for levomethorphan. The analytical target profile was defined as a method that should be able to determine levomethorphan with acceptable precision and accuracy at the 0.1 % level. From initial scouting experiments, a dual selector system consisting of sulfated β‐cyclodextrin and methyl‐α‐cyclodextrin was identified. The critical process parameters were evaluated in a fractional factorial resolution IV design followed by a central composite face‐centered design and Monte Carlo simulations for defining the design space of the method. The selected working conditions consisted of a 30/40.2 cm, 50 μm id fused‐silica capillary, 30 mM sodium phosphate buffer, pH 6.5, 16 mg/mL sulfated β‐cyclodextrin, and 14 mg/mL methyl‐α‐cyclodextrin at 20°C and 20 kV. The method was validated according to ICH guideline Q2(R1) and applied to the analysis of a capsule formulation. Furthermore, the apparent binding constants between the enantiomers and the cyclodextrins as well as complex mobilities were determined to understand the migration behavior of the analytes.     

Evaluation of balhimycin as a chiral selector for enantioresolution by capillary electrophoresis

The glycopeptide antibiotic balhimycin and its haloanalogue bromobalhimycin were evaluated as chiral selectors for enantioresolution by capillary electrophoresis. In order (i) to eliminate the adsorption of the glycopeptide antibiotics on the capillary wall, (ii) to shorten the separation time and (iii) to improve the detection sensitivity, a combined approach of the dynamic surface coating technique, the co-electroosmotic flow electrophoresis technique and the partial filling technique was employed for the enantioresolution of 16 acidic racemates. The effect of experimental parameters (plug length of the partial filling solution containing the chiral selector, selector concentration and buffer pH) on enantiorecognition was investigated. Furthermore, the enantiorecognition ability imparted by balhimycin, bromobalhimycin and vancomycin were compared. For most tested compounds, the highest enantiorecognition was obtained with balhimycin as chiral selector. Only in the case of the enantioresolution of tiaprofenic acid, vancomycin showed a superior enantiorecognition.     

Development of a validated capillary electrophoresis method for enantiomeric purity testing of dexchlorpheniramine maleate

A capillary zone electrophoresis method has been developed for the detection of 0.1% of (R)-levochlorpheniramine maleate in samples of (S)-dexchlorpheniramine maleate. Using 1.5 mM carboxymethyl-beta-cyclodextrin in an acidic background electrolyte, resolution values of more than 10 were obtained. Under these conditions the R-enantiomer is migrating in front of the bulk S-enantiomer. The assay was validated for linearity (2-10 microg/ml; R2 = 0.9992), selectivity [(RS)-pheniramine maleate and (RS)-brompheniramine maleate], limit of detection (0.25 microg/ml), limit of quantification (0.75 microg/ml), analytical precision (intra- and inter-day variability), repeatability of the method (RSD = 5.0%) and accuracy. In samples of dexchlorpheniramine maleate from two different manufacturers, concentrations of, respectively, 0.15% and 1.95% (m/m) of levochlorpheniramine maleate were detected. The method was compared to the HPLC method described in the European Pharmacopoeia III monograph.     

Design and synthesis of coumarin substituted oxathiadiazolone derivatives having anti-inflammatory activity possibly through p38 MAP kinase inhibition

Compounds containing oxathiadiazolone nucleus bearing substituted coumarin ring were designed and synthesized while retaining the pharmacophores required for binding with p38 MAP kinase. A four-step synthetic scheme was employed for the synthesis of 7-methoxy-4-(3t?-substituted-2t?-oxo-1t?,2t?,3t?,5t?-oxathiadiazol-4t?-yl)-coumarin. The reactions were monitored by TLC and structures of the intermediates and the target compounds were ascertained by IR, NMR, Mass spectral data. The compounds were found to possess anti-inflammatory activity comparable to indomethacin. Superimposition studies of the target compounds with the lead p38 kinase inhibitors suggested that anti-inflammatory activity of the target compounds may be due to p38 MAP kinase inhibition. It was also suggested that methoxy group on coumarin nucleus may improve the binding profile with p38 MAP kinase.     

Enantiomeric purity determination of propranolol by capillary electrophoresis using dual cyclodextrins and a polyacrylamide-coated capillary.

The use of a chirally selective capillary electrophoresis method is reported for the enantioselective purity determination of propranolol drug substance. The method employed a combination of both charged and neutral cyclodextrin. An internally coated capillary was used to suppress electroosmotic flow and potential peak tailing. The method was capable of monitoring below 0.1% m/m of the undesired impurity. Acceptable validation data was also obtained for recovery, linearity, and for both short and long-term injection precision.     

Comparative studies of various run buffers for chiral capillary electrophoresis using chiral crown ether as a chiral selector

In the capillary electrophoretic separation of primary amine enantiomers using (+)-(18-crown-6)-tetracarboxylic acid (18C6H4) as a chiral selector, the presence of run buffer constituents such as tris(hydroxymethyl)aminomethane (Tris) or Na+ competing with analytes for 18C6H4, diminishes the effectiveness of 18C6H4. In order to determine appropriate buffer systems for 18C6H4, various run buffer cationic components including Tris, 1,3-bis[tris(hydroxymethyl)methylamino]propane, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, triethanolamine, tetramethylammonium, and Na+ were compared. Quantitative studies of the effects of the competitive constituents were carried out by measuring the electrophoretic mobilities of histidine as a function of the 18C6H4 concentration. We also derived a simple equation to estimate the optimal chiral selector concentration for a maximum mobility difference in the presence of a competitive inhibitor.     

Determination of enantiomeric purity of a novel COX-2 anti-inflammatory drug by capillary electrophoresis using single and dual cyclodextrin systems

E-6087 is the most advanced compound among the cyclooxygenase-2 (COX-2) inhibitor drugs developed in our company. Its activity is mainly associated with the S(-)-enantiomer (E-6232), whereas the R(-)-enantiomer (E-6231) becomes an impurity whose content should be determined. Five main impurities and degradation products of E-6232 have been found (E-6144, E-6024, E-6072, E-6397 and E-6132), and some of them co-elute with the distomer when using a chiral high-performance liquid chromatography (HPLC) method. Consequently, we have optimized the separation of all the impurities from the two enantiomers of E-6087 by capillary electrophoresis (CE), in order to use the method for the enantiomeric purity determination of E-6232. The effect of the methanol (MeOH) content in the background electrolyte (BGE), the sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) and heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD) concentration, and the capillary temperature have been studied. Separation of all compounds could be achieved in different systems, either in a single CD-system (with SBE-beta-CD) or in a dual CD-system (with DM-beta-CD as a neutral CD). By using the dual CD system a limit of detection (LOD) and a limit of quantitation (LOQ) of 0.03% and 0.1% of distomer, respectively, were achieved*.     

Quality by design-assisted development of a capillary electrophoresis method for the enantiomeric purity determination of tenofovir

A CE method was developed and validated for the assessment of the chiral purity of the drug tenofovir applying a quality by design approach. Following selection of a quaternary ammonium β-CD as chiral selector, a fractional factorial resolution V+ design was employed for identification of the critical process parameters, while a central composite design served for method optimization. The final method used a 40/50.2 cm, 50 μm id fused-silica capillary, a BGE composed of a 100 mM sodium phosphate buffer, pH 6.4, containing 45 mg/mL quaternary ammonium β-CD, an applied voltage of 18 kV, and a capillary temperature of 22°C. Robustness was assessed by a Plackett–Burman design. The method was validated according to guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and enabled the determination of the (S)-enantiomer of tenofovir at the 0.1% level.     

Enantiorecognition of profens by capillary electrophoresis using a novel chiral selector eremomycin

The evaluation of a macrocyclic glycopeptide antibiotic, eremomycin, as a chiral selector in capillary electrophoresis (CE) has been performed. The stability of eremomycin in solution and capillary electrolyte, as well as its optical and electrophoretic properties have been discussed. The effect of experimental parameters influencing the enantioseparation of several profens has been studied. Excellent enantioseparation of profens has been achieved and migration order has been validated. Comparison of enantioseparations of profens in CE by using eremomycin-mediated electrolytes and in HPLC with eremomycin immobilized on silica has revealed similar trends for both methods.     

Application of charged single isomer derivatives of cyclodextrins in capillary electrophoresis for chiral analysis

The review focuses on the role of ionic or ionisable single isomer derivatives (SIDs) of cyclodextrins on the separation of chiral analytes in capillary electrophoresis (CE), covering the period since the year 2000. The advantages of using pure compounds are discussed, as well as the ways to optimise the separations in the context of a rational approach to these techniques. Specific attention is paid to the modulation of the selector–analyte interaction. The advantage due to a detailed knowledge of equilibria occurring in solution during the CE run is underlined, particularly in the case of the presence of metal complexes, as occurs in chiral ligand exchange capillary electrophoresis (CLECE).