Enabling chiral separations in discovery chemistry with open‐access chiral supercritical fluid chromatography

Work from this paper details a novel walk‐up open‐access (OA) approach to enable chiral analytical method development and preparative separation of enantiomers in early discovery chemistry using supercritical fluid chromatography (SFC). We have demonstrated the success of this OA approach using immobilized chiral stationary phases (CSPs). After screening a diverse set of racemic drug candidates, we have concluded that a simplified OA chiral SFC platform can successfully purify approximately 60% of the analysed racemates. This streamlined OA workflow enables medicinal chemists with limited expertise in chiral method development to successfully and rapidly purify enantiomers for their projects using Waters UPC² and Prep100‐SFC instrumentation.     

Large scale chiral chromatography for the separation of an enantiomer to accelerate drug development

There are several approaches to produce enantiomerically pure drug substances, such as recrystallization, catalytic process (ligand and enzyme), indirect chromatographic resolution, and direct chromatographic resolution. However, the use of preparative chromatography with chiral stationary phases seems to be most effective for early phase projects, where the time and resources on the developments need to be minimized to get the drug candidates into the clinical studies. We showed that by following a well-defined process, chiral chromatography can be easily scaled up from an analytical system to a pilot plant system. We also used the results from a multicolumn continuous chromatography (MCC) study to conclude that MCC can be a cost-effective production method for chiral manufacturing.     

Studies on the racemization of a stereolabile 5-aryl-thiazolidinedione

The enantiomers of the stereolabile peroxisome proliferator-activated receptor (PPAR) agonist, 1, were isolated by preparative chiral chromatography and their absolute configuration established using a combination of chromatographic and NMR methods. Enantiomer interconversion was investigated under a variety of conditions, with rapid racemization being observed in most solvents, including all aqueous systems studied, irrespective of pH. Rapid racemization in both dog and human plasma was confirmed by chiral HPLC with MS detection.     

Preparative chiral chromatographic resolution of enantiomers in drug discovery

A brief account is given of the role of preparative chromatography for the direct separation of enantiomers in the drug discovery process. Although it is not yet possible to predict the outcome of a chromatographic resolution attempt, and the optimisation procedure sometimes might be time-consuming, the technique is still indispensable as a means to obtain both enantiomers in pure form from a drug racemate for biological testing. The most suitable types of chiral stationary phases (CSPs) available for this purpose are discussed with special reference to loadability and compatibility with different mobile phase systems.     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

Immobilized horse liver alcohol dehydrogenase as an on‐line high‐performance liquid chromatographic enzyme reactor for stereoselective synthesis

Horse liver alcohol dehydrogenase (HLADH) has been non‐covalently immobilized on an immobilized artificial membrane (IAM) high‐performance liquid chromatography (HPLC) stationary phase. The resulting IAM‐HLADH retained the reductive activity of native HLADH as well as the enzyme's enantioselectivity and enantiospecificity. HLADH was also immobilized in an IAM HPLC stationary phase prepacked in a 13 × 4.1 mm ID column to create an immobilized enzyme reactor (HLADH‐IMER). The reactor was connected through a switching valve to a column containing a chiral stationary phase (CSP) based upon p‐methylphenylcarbamate derivatized cellulose (Chiralcel OJR‐CSP). The results from the combined HLADH‐IMER/CSP and chromatographic system demonstrate that the enzyme retained its activity and stereoselectivity after immobilization in the column and that the substrate and products from the enzymatic reduction could be transferred to a second column for analytical or preparative separation. The combined HLADH‐IMER/CSP system is a prototype for the preparative on‐line use of cofactor‐dependent enzymes in large‐scale chiral syntheses. Chirality 11:39–45, 1999. © 1999 Wiley‐Liss, Inc.     

Axially chiral Ni(II) complexes of α‐amino acids: Separation of enantiomers and kinetics of racemization

Herein we present design, synthesis, chiral HPLC resolution, and kinetics of racemization of axially chiral Ni(II) complexes of glycine and di‐(benzyl)glycine Schiff bases. We found that while the ortho‐fluoro derivatives are configurationally unstable, the pure enantiomers of corresponding axially chiral ortho‐chloro‐containing complexes can be isolated by preparative HPLC and show exceptional configurational stability (t_1/2 from 4 to 216 centuries) at ambient conditions. Synthetic implications of this discovery for the development of new generation of axially chiral auxiliaries, useful for general asymmetric synthesis of α‐amino acids, are discussed.     

Preparative scale enantioseparation of a chiral epoxide: Comparison of liquid chromatography and simulated moving bed adsorption technology

The feasibility of using simulated moving bed technology (SMB) for chiral separation on cellulose triacetate is demonstrated on the preparative scale: 1 kg of a chiral epoxide has been separated. On comparing SMB technology with conventional liquid chromatography it turns out that the main advantage of SMB lies in the significant reduction of mobile phase consumption. The process design for SMB is made theoretically and the predictions are confirmed by our pilot study. © 1993 Wiley-Liss, Inc.     

Preparation of chiral building blocks and auxiliaries by chromatography on cellulose triacetate (CTA I): Indications for the presence of multiple interaction sites in CTA I

Due to the increasing demand for optically active compounds, the development of methods supplying optically pure isomers is intensively progressing. Among these methods the chromatographic resolution on chiral stationary phases is very promising, although only a limited number of preparative applications have been reported so far. In this work, we demonstrate that especially cellulose triacetate I (CTA I) as a chiral phase presents a number of advantages for this purpose. The broad applicability and the high loading capacity of CTA I are particularly important features for preparative chromatography. Nevertheless, slight structural modifications of the racemates to be resolved can often strongly improve the resolution. This strategy has been applied to numerous practical problems and is illustrated in this work taking as examples some chiral building blocks and auxiliaries. Moreover, a systematic investigation of the influence of a substituent in the para-position of the phenyl ring for different series of aromatic compounds led to the conclusion that a large number of different interaction sites must be present in the chiral environment of CTA I.     

Recent advances in methods of direct optical resolution

Very great advances have been made in the field of direct optical resolution of organic compounds by chromatographic techniques. Chiral capillary gas chromatography now permits a determination of the enantiomeric composition of a few nanograms of a compound present in a mixture of many others. Coupled with high resolution mass spectrometry the technique will additionally permit structural elucidation; of great interest in pheromone research and related areas. Analytical separations of enantiomers are now also carried out by high-performance liquid chromatography (HPLC) methods based on a variety of principles. Basically, two main types are used, differing as to whether the mobile phase has to be a chiral medium or not. Two-dimensional HPLC, whereby compounds separated on a non-chiral column are progressively and automatically transferred to a chiral column for optical resolution, has been used successsfully for chiral amino acid separations. Many different chiral sorbents for preparative LC and HPLC resolutions have been prepared; some of these are now used in columns capable of producing pure enantiomers from a given racemate at a rate of the order of one gram/hour in continuous, automatic HPLC procedures. Apart from all important applications of these results of optical resolution technology, an increased knowledge of the underlying chiral recognition phenomena responsible for enantioselection has also been achieved.     

Preparative separation of nebivolol isomers by improved throughput reverse phase tandem two column chromatography

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)⁻¹ and productivity of 20 g L⁻¹ day⁻¹.     

Evaluation of π-acid chiral stationary phases deriving from tyrosine and related amino acids for the chromatographic resolution of racemates: Specific requirements for enantiorecognition ability

Chromatographic applications of three novel chiral stationary phases (CSPs) deriving from (S)-(N)-(3,5-dinitrobenzoyl)tyrosine are reported, under liquid chromatographic (LC) and subscritical fluid chromatographic (SubFC) conditions. Two grafting modes of the chiral moiety have been experimented starting either from γ-mercaptopropyl-silanized (type 1) or γ-aminopropyl-silanized (type 2) silica gels. For type 2 CSPs an evaluation of the stability of the amide linkage was achieved by means of SubFC; the relative contriution of ionic and covalent bindings to the ciral recognitio aility was then outlined. The chromatographic properties of these CSPs were compared with those of the corresponding CSPs deriving from phenylglycine, p-hydroxyphenylglycine, and phenylalanine for the resolution of some tertiary phosphine oxide, naphthoyl amide, and α-methylene γ-lactam enantiomers. Some simple requirements regarding the solute and CSP structures for chiral recognition ability can be inferred from these results. In addition, the resolutio of π-acid α-N-(3,5-dinitrobenzoyl)amino esters was investigated on these π-acid CSPs. An example of preparative scale chromatography is also presented.     

Chiral separation of tryptophan enantiomers by liquid chromatography with BSA-silica stationary phase

The separation of tryptophan enantiomers was carried out with medium-pressure liquid chromatography using BSA (bovine serum albumin)-bonded silica as a chiral stationary phase. The influence of various experimental factors such as pH and ionic strength of mobile phase, separation temperature, and the presence of organic additives on the resolution was studied. In order to expand this system to preparative scale, the loadability of sample and the stability of stationary phase for repeated use were also examined. The separation of tryptophan enantiomers was successful with this system. The data indicated that a higher separation factor (α) was obtained at a higher pH and lower temperature and ionic strength in mobile phase. Addition of organic additives (acetonitrile and 2-propanol) in mobile phase contributed to reduce the retention time of L-tryptophan. About 30% of the separation factor was reduced after 80 days of repeated use.     

Chiral HPLC method for chiral purity determination of paroxetine drug substance

This article describes a chiral HPLC method for (+) trans isomer of paroxetine in a paroxetine drug substance. The method development was performed to establish a suitable HPLC system in order to separate both enantiomers. It was found that a system based on a Chiralpak AD column was suitable for the analysis. Proper column maintenance and the optimized eluent composition allowed good reproducibility and sensitivity for the method. The method was also checked on a number of different columns using different HPLC equipment and gave both reproducible chromatography and reproducible quantitative results.     

Preparative chromatographic resolution of racemates using HPLC and SFC in a pharmaceutical discovery environment

The preparative chromatographic resolution of racemates has become a standard approach for the generation of enantiomers in pharmaceutical discovery laboratories. This paper will discuss the use of preparative HPLC and SFC to generate individual enantiomers for discovery activities. Analytical HPLC and SFC method development to rapidly screen chiral stationary phases and solvent combinations will be presented. The usefulness of preparative chromatographic resolution of racemates will be demonstrated through the presentation of numerous non-routine case studies from the laboratories at Amgen.     

Chiral counter-current chromatography: A survey of its instrument,mechanism, procedure,and applications

The chiral separation by counter-current chromatography has made great progress in the past three decades. It has become increasingly popular in the field of chiral separation, and many applications have been introduced during the last years. This review mainly focuses on the current topics, applications, and trends in chiral separation by counter-current chromatography. It contains the development of modern counter-current chromatography apparatus, theory of counter-current chromatography, overview of applications of chiral counter-current chromatography enantioseparation, its current situation, and challenges. At last, some conclusions and perspectives also have been discussed in this review.     

A novel design of simulated moving bed (SMB) chromatography for separation of ketoprofen enantiomer

A simulated moving bed (SMB) chromatography system is a powerful tool for preparative scale separation, which can be applied to the separation of chiral compound. We have designed our own lab-scale SMB chromatography using 5 HPLC pumps, 6 stainless steel columns and 4 multi-position valves, to separate a racemic mixture of ketoprofen in to its enantiomers. Our design has the characteristics of the low cost for assembly for the SMB chromatography and easy repair of the unit, which differs from the designs suggested by other investigators. It is possible for the flow path through each column to be independently changed by computer control, using 4 multi-position rotary valves and 5 HPLC solvent delivery pumps. In order to prove the operability of our SMB system, attempts were made to separate the (S)-ketoprofen enantiomer from a ketoprofen racemic mixture. The operating parameters of the SMB chromatography were calculated for ketoprofen separation from a batch chromatography experiment as well as by the triangle theory. With a feed concentration of 1 mg/mL, (S)-ketoprofen was obtained with a purity of 96% under the calculated operating conditions.     

Separation of Nadolol Stereoisomers by Chiral Liquid Chromatography at Analytical and Preparative Scales

The separation of the four nadolol stereoisomers on Chiralpak® AD by chiral liquid chromatography was carried out at both analytical and preparative scales. A screening of possible mobile‐phase compositions was performed using different alcohol–hydrocarbon mixtures. The results obtained confirm the use of 20:80:0.3 ethanol‐hexane‐diethylamine reported by McCarthy (1994) but introduce other possibilities for the complete resolution of the four nadolol stereoisomers at analytical scale, namely, the mixtures 30–40:70–60:0.3 ethanol‐heptane‐diethylamine. Additionally, this work describes how retention and resolution depend on the ethanol content in hexane and heptane mixtures. The separation of nadolol stereoisomers is also carried out at preparative scale and different alcohol–hydrocarbon compositions are proposed, depending on the target component to be obtained. Particularly, this work presents the experimental separation of the more retained nadolol stereoisomer (RSR‐nadolol) by simulated moving bed (SMB) chromatography using an 80:20:0.3 ethanol‐heptane‐diethylamine mobile phase. For a 2 g/l feed concentration, RSR‐nadolol is 100% recovered at the extract outlet stream, 100% pure, and with a system productivity of 0.65 g_{RSR‐nadolol}/(l_bed^.h) and a solvent consumption of 9.6 l_solvent/g_{RSR‐nadolol}. Chirality 25:197–205, 2013. © 2013 Wiley Periodicals, Inc.     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

Excavations in drug chirality: 1. Cyclothiazide

There is a great deal of current interest in the role and importance of chirality in the development of new drugs, but little attention is being paid to the stereochemistry of older drugs. Indeed, many older chiral drugs were introduced without adequate information on their stereochemical identity or composition. We have examined one such drug, the antihypertensive diuretic agent cyclothiazide. Standard sources of drug information and the research literature do not provide data on the stereochemical composition of clinically used cyclothiazide, although scattered reports indicate that the drug may consist of "several stereoisomers." Inspection of the chemical structure of the drug, 6-chloro-3,4-dihydro-3-(5-norbornen-2-yl)-2H-1,2,4-benzothiadiazin e-7- sulfonamide 1,1-dioxide, shows that it can exist as eight stereoisomers that may form four racemates. Using synthesis, fast-atom-bombardment mass spectrometry, gas-liquid chromatography, chiral and nonchiral high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, we determined that pharmaceutical cyclothiazide is in fact a mixture of the eight stereoisomers in the form of the four racemates. The two racemates with endo configuration at the norbornene moiety predominate over the exo racemates, and small but significant differences in isomer distribution between different batches of the drug were observed. We urge that in studies of older drugs the stereochemical details be considered.