V. Scott Sharp

Evaluation of a new macrocyclic antibiotic as a chiral selector for use in capillary electrophoresis

Abstract A new macrocyclic antibiotic, LY307599, has been evaluated as a chiral selector for the separation of the enantiomers of flurbiprofen using capillary electrophoresis (CE). The effect of varying separation buffer parameters such as buffer strength, pH, LY307599 concentration and methanol concentration were assessed. Using the optimized CE conditions, the separation of flurbiprofen enantiomers can be achieved using LY307599 as a chiral selector.

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high-performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pKa values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids.

ENANTIOMERIC SEPARATION OF AN AMPA ANTAGONIST USING A CHIROBIOTIC T™ COLUMN WITH HPLC AND EVAPORATIVE LIGHT-SCATTERING DETECTION

A Chirobiotic T™ column was used for the direct separation of AMPA receptor antagonist LY293558 and the undesired enantiomer LY293559 in bulk drug substance. High performance liquid chromatography (HPLC) separation of the enantiomers was optimized using reversed phase and hydrophilic interaction chromatography (HILIC) by varying the organic composition of the mobile phase. Baseline resolution was achieved allowing accurate, trace level quantitation of the undesired enantiomer in the optically pure bulk material. Because the analytes lack a sufficient ultraviolet chromophore, an evaporative light-scattering detector (ELSD) was used to enhance detection. The ELSD was capable of obtaining detection limits as low as 0.1% of the undesired enantiomer. Additional experiments were conducted to assess the linearity, precision, and accuracy of the HPLC-ELSD system.

Evaluation of a New Pepsin Enzyme Chiral Stationary Phase for the Optimized Separation of Seproxetine (S-Norfluoxetine) from R-Norfluoxetine

Abstract A thorough analysis of a new commercially available pepsin chiral stationary phase (CSP) has been completed using seproxetine (S-norfluoxetine) hydrochloride bulk drug substance and R-norfluoxetine hydrochloride as the test analytes. Chromatographic properties of this new Ultron ES-Pepsin column were investigated by varying key mobile phase parameters (pH, flow rate, buffer strength and organic concentration), column temperature and sample loading. After observing and plotting changes in retention, resolution and theoretical plates based on corresponding variation in these parameters, it is possible to choose conditions for the separation that are optimum and robust. The subsequent method validation demonstrated acceptable precision, linearity, recovery, selectivity, limit of detection and ruggedness for the determination of R-norfluoxetine in seproxetine hydrochloride bulk drug substance.

High performance liquid chromatographic enantioseparation development and analytical method characterization of the carboxylate ester of evacetrapib using an immobilized chiral stationary phase with a non-conventional eluent system.

Using multiple HPLC chromatographic modes and various chiral columns in the context of an automated screening system, a potential separation was initially identified for the methyl ester of evacetrapib and its stereoisomers using an immobilized polysaccharide-based HPLC column. The bonded nature of this column, the Chiralpak(®) IC, allows for enhanced separation development with a diverse solvent range not amenable to standard coated chiral stationary phases. The ternary eluent system ultimately identified provided isomer resolutions not obtainable via the more established hexane/alcohol or polar organic chromatographic modes. A systematic separation development process is described, first for the resolution of the isomers, and later incorporating five potential impurities. A robust separation system was eventually developed that effectively resolves all compounds within a reasonable analysis time.

Evaluation of an HPLC Chiral Separation Flow Scheme for Small Molecules

Abstract An HPLC chiral separation flow scheme was developed for identification of suitable enantioseparation conditions for small molecules. This flow scheme employs various chiral stationary phases (CSPs) and separation modes with the aim of improving efficiency by leading the scientist toward a reliable enantioseparation through a reduced number of experiments. In cases where a partial chiral separation is achieved with a particular CSP, guidance in the flow scheme is provided to improve resolution. Using prior knowledge, literature references, and data from 60 nonproprietary compounds analyzed in this study, the flow scheme was developed with separation mode (solvent compatibility) versatility in mind.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Mercks MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

Stereoselective high-performance liquid chromatography and analytical method characterization of evacetrapib using a brush-type chiral stationary phase: a challenging isomeric separation requiring a unique eluent system.

Using HPLC chiral separation screening, various columns representing the polysaccharide, macrocyclic antibiotic and brush classes were assessed in multiple chromatographic modes for the separation of evacetrapib, a potential cardiovascular drug, from its enantiomer, two diastereomers and several impurities. Screening data consistently pointed to the brush-type Whelk-O 1 chiral column as most promising for this task. A systematic separation optimization process is outlined using the (S,S) Whelk-O 1 chiral column, first for the resolution of the isomers, and later including six potential impurities. A relatively complex yet rugged separation system was eventually identified that effectively resolves all compounds within a reasonable analysis time, and should serve as an adequate tool for evacetrapib bulk drug enantiopurity measurement.

Investigation of the Mechanism of Racemization of Litronesib in Aqueous Solution: Unexpected Base‐Catalyzed Inversion of a Fully Substituted Carbon Chiral Center

Mitosis inhibitor (R)-litronesib (LY2523355) is a 1,3,4-thiadiazoline-bearing phenyl and N-(2-ethylamino)ethanesulfonamido-methyl substituents on tetrahedral C5. Chiral instability has been observed at pH 6 and above with the rate of racemization increasing with pH. A positively charged trigonal intermediate is inferred from the fact that p-methoxy substituent on the phenyl accelerated racemization, whereas a p-trifluoromethyl substituent had the opposite effect. Racemization is proposed to occur through a relay mechanism involving intramolecular deprotonation of the sulfonamide by the side chain amino group and attack of the sulfonamide anion on C5, cleaving the C5S bond, to form an aziridine; heterolytic dissociation of the aziridine yields an ylide. This pathway is supported by (1) a crystal structure providing evidence for a hydrogen bond between the sulfonamide NH and the amino group, (2) effects of substituents on the rate of racemization, and (3) computational studies. This racemization mechanism results from neighboring group effects in this densely functionalized molecule. Of particular novelty is the involvement of the side-chain secondary amino group, which overcomes the weak acidity of the sulfonamide by anchimeric assistance.

Development of Preparative Chiral Separations Using an Intelligent Chiral Resolution System

Abstract Chiral preparative chromatography is a technology increasingly used in the pharmaceutical industry to deliver enantiomerically pure drug candidates. A strategy for rapid screening of conditions on polysaccharide chiral stationary phases (CSPs) has been developed with an emphasis on preparative applications. Sample solubility is a major limiting factor in preparative chromatography. Most reported analytical chiral separations on polysaccharide CSPs are with eluent systems consisting of mainly hexane and small amounts of alcohol as a modifier. The intelligent chiral resolution system (ICRS) was developed to emphasize polar solvents as the first choice for eluent systems. Examples of this approach, and details on the screening procedure are presented. Using the system, chiral preparative procedures can be developed for most compounds in approximately 3 hours.