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.     

Strategic use of preparative chiral chromatography for the synthesis of a preclinical pharmaceutical candidate

The modern use of preparative chromatography in pharmaceutical development is illustrated by the case of a recent preclinical candidate from these laboratories. The synthesis of the candidate employed a coupling of two enantiopure intermediates, each of which could be resolved using preparative chiral chromatography. SFC screening was employed to identify the enantioselective stationary phases, and semipreparative SFC methods derived from this screening were used to produce gram amounts of enantiopure intermediate for initial studies. However, initial larger scale resolution required the translation of the SFC methods to HPLC conditions. Preparative chiral HPLC on a 30-cm i.d. column was then used to produce enantiopure intermediates which were coupled to give 170 g of the preclinical candidate. Subsequent preparation of the candidate at larger scale for later-stage clinical evaluation employed an improved synthesis in which one component was constructed by asymmetric synthesis. Resolution of the other component, now a more advanced intermediate, was carried out using newly obtained large-scale SFC equipment. Some discussion is presented on the varying strategies whereby preparative chiral chromatography can be used to support either short-term or long-term synthetic goals in preclinical pharmaceutical development.     

The application of preparative batch HPLC, supercritical fluid chromatography, steady-state recycling, and simulated moving bed for the resolution of a racemic pharmaceutical intermediate

This article discusses the chromatographic resolution of a racemic pharmaceutical intermediate. Preparative batch high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), steady-state recycling (SSR), and simulated moving bed (SMB) were used to resolve a total of 12.2 kg of a racemic pharmaceutical intermediate. In this study, a first batch of 0.8 kg of racemate was separated on the preparative batch HPLC and SFC, and subsequently another 5.9 kg of racemate was separated on the SSR. Lastly, a third batch of 5.5 kg was separated on the SMB. The separation conditions and results of these techniques are discussed. The productivities and solvent costs of SFC versus HPLC are compared. The productivities and solvent costs of SMB, SSR, and HPLC are also compared. The analytical method development and process optimization of these processes are also discussed in this article.     

Effects of Hexane in Supercritical Fluid Chromatography for the Separation of Enantiomers

Supercritical fluid chromatography (SFC), operated in conventional mode, is normally recognized as normal phase chromatography, and uses a solvent combination of supercritical CO₂ and alcohols to separate compounds. Hexane, a commonly used solvent in normal phase liquid chromatography (NP‐LC), is rarely used in SFC and, in some cases, is added to the organic modifiers to increase liquid content in order to achieve better efficiency in preparative SFC for poorly retained compounds. Although hexane is believed to have similar solvent strength to that of supercritical CO₂, its effects on the enantioseparation in SFC is largely unknown. To understand the chromatographic effects of an apolar solvent, such as hexane in SFC, we compared the chromatographic behaviors of 35 chiral compounds using a parallel SFC method under traditional SFC mode of only “pure” alcohol‐CO₂ to that of hexane‐assisted SFC (HA‐SFC), which uses mixtures of alcohol and hexane (as cosolvents) and CO₂. We observed that, in some cases, hexane behaves just like supercritical CO₂, where replacement of a portion of CO₂ with hexane does not significantly change retention times or resolution of the peaks. In many cases, however, addition of hexane in mobile phases does affect chromatographic behavior of one or both enantiomers. Such effects might provide opportunities for separation of some enantiomers. Chirality 28:192–198, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Preparative chiral chromatography and chiroptical characterization of enantiomers of omeprazole and related benzimidazoles

To chiroptically characterize the enantiomers of omeprazole and some structurally related benzimidazoles with circular dichroism (CD), preparative chiral liquid chromatography was utilized for the isolation of the pure enantiomers. A limited analytical column screen was performed identifying Kromasil-CHI-TBB and the amylose-based phases Chiralpak AD and AS as possible chiral stationary phases (CSPs) for the preparative scale separation of the enantiomers of the different benzimidazoles. Optimization of the chromatographic conditions with respect to retention, enantioseparation, and resolution was achieved by variation of the mobile phase constituents as well as of temperature. Because of the lability of the compound in slightly acidic media, supercritical fluid chromatography (SFC) could not be applied for a preparative scale separation of the enantiomers. The separation of omeprazole was optimized to give high throughput (2.6 kg racemate/kg CSP/day) and high enantiomeric excess of the obtained isomers. The absolute configurations of the pure enantiomers of rabeprazole, lansoprazole, and pantoprazole were determined from the strong correlation to the CD spectrum of (+)-(R)-omeprazole. For all the compounds, the (+)-enantiomers displayed similar chiroptical features as (+)-(R)-omeprazole and were thus assigned the (R)- configuration. Elution order of the optical isomers was monitored by injecting racemic solutions spiked with one of the isomers and also by an on-line laser polarimeter. Both the type of CSP and also the mobile phase constituents had a strong effect on elution order of the enantiomers.     

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.     

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.     

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.     

Some applications of chiral liquid affinity chromatography using bovine serum albumin as a stationary phase

The enantioselectivity excerted by many proteins can be utilized for direct optical resolution in liquid chromatographic processes whereby the protein is used as a stationary phase. Bovine serum albumin (BSA), covalently bound to a suitable support, has been shown to act as a chiral discriminator for a variety of racemic organic compounds in aqueous buffers. Columns packed with BSA-silica can be used for determination of enantiomeric composition in aqueous solvents at very low concentrations by HPLC. This technique opens up new possibilities for the preparative isolation of micrograms amounts of enantiomers and for studies of stereoselectivity and mechanisms in enzymatic and microbial reactions.     

Challenges and frustrations in the separation and analysis of chiral agrochemicals

The development of chiral HPLC methods and isolation techniques within Zeneca Agrochemicals (formerly ICI Agrochemicals) is reviewed. The use of low temperature to improve chiral separations has been successfully applied to production analysis, but although useful for some compounds it is regrettably not a universal panacea for all poor separations. The need to isolate small quantities of individual enantiomers from new compounds for research evaluation has led us to devise a more universal and cheap chiral stationary phase (CSP) for Preparative-LC. Joint academic research produced a CSP based on tartaric acid which was made commercially available and it was gratifying to find it was the only phase able to resolve a novel insecticide. However, as new CSPs emerged almost every month, our attention turned to using a universal chiral detector for analysis, rather than via separation of individual enantiomers. Diode laser-based polarimeters offered the opportunity of cheap, sensitive chiroptical detectors for HPLC and the ability to move away from chiral columns in both research and production analysis. Jointly sponsored research with a university has successfully explored the versatility of chiroptical detectors in agrochemical and food analysis. Comparison of chiral SFC with chiral HPLC and an extensive evaluation of established and research agrochemicals on a wide range of commercial CSPs have led to a revised method development strategy. Current work with high load displacement chiral chromatography will be described as a potential means of isolating pure enantiomers from racemates. © 1994 Wiley-Liss, Inc.     

Enantioseparation of the Six Important Intermediates of Homoharringtonine With Immobilized Cellulose Chiral Stationary Phase

A new liquid chromatographic method has been developed for the chiral separation of the enantiomers of intermediates in the preparation of the ester side‐chain of homoharringtonine. The enantiomers were separated by a Chiralpak IC (250 × 4.6 mm, 5 µm) in normal phase high‐performance liquid chromatography (HPLC). Four compounds were baseline resolved. By comparing the chromatographs of racemates and single enantiomers of the six intermediates, the enantiomeric excess values of the single enantiomers were evaluated, and the elution orders of the enantiomers were established. Chirality 27:538–542, 2015. © 2015 Wiley Periodicals, Inc.     

Predictability of Enantiomeric Chromatographic Behavior on Various Chiral Stationary Phases Using Typical Reversed Phase Modeling Software

Pharmaceutical companies worldwide tend to apply chiral chromatographic separation techniques in their mass production strategy rather than asymmetric synthesis. The present work aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab HPLC method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability: macrocyclic antibiotics‐based columns (Chirobiotic V and T), polysaccharide‐based chiral column (Chiralpak AD‐RH), and protein‐based chiral column (Ultron ES‐OVM). Preliminary basic runs were implemented, then exported to DryLab after peak tracking was accomplished. Prediction of the effect of % organic mobile phase on separation was possible for separations on Chirobiotic V for several probes: racemic propranolol with 97.80% accuracy; mixture of racemates of propranolol and terbutaline sulphate, as well as, racemates of propranolol and salbutamol sulphate with average 90.46% accuracy for the effect of percent organic mobile phase and average 98.39% for the effect of pH; and racemic warfarin with 93.45% accuracy for the effect of percent organic mobile phase and average 99.64% for the effect of pH. It can be concluded that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory. Chirality 25:506–513, 2013. © 2013 Wiley Periodicals, Inc.     

HPLC and SFC enantioseparation of (±)‐Corey lactone diol: Impact of the amylose tris‐(3,5‐dimethylphenylcarbamate) coating amount on chiral preparation

As an important intermediate of prostaglandins and entecavir, optically pure Corey lactone diol (CLD) has great value in the pharmaceutical industry. In this work, the enantioseparation of (±)‐CLD was evaluated using high‐performance liquid (HPLC) and supercritical fluid chromatography (SFC). In HPLC, the separations of CLD enantiomers on polysaccharide‐based chiral stationary phases with both normal phase and polar organic phase were screened. And the conditions for the enantioseparation were optimized in HPLC and SFC, including the selection of mobile phase, temperature, back‐pressure, and other conditions. More important, it was found that the chiral resolutions were greatly enhanced by the increase of the coating amount of ADMPC (amylose tris‐(3,5‐dimethylphenylcarbamate)) under both HPLC and SFC conditions, which can lead to the increase of the productivity and the decrease of the solvent consumption. The preparations of optically pure CLD were evaluated on a semi‐preparative (2 × 25 cm) column packed with 30% ADMPC‐coated CSP under HPLC and SFC conditions. Preparative performances in terms of kkd are 1.536 kg racemate/kg CSP/day and 1.248 kg racemate/kg CSP/day in HPLC and SFC, respectively.     

Synthesis,HPLC Enantioresolution,and X‐ray Analysis of a New Series of C5‐methyl Pyridazines as N‐Formyl Peptide Receptor (FPR) Agonists

The synthesis of three racemates and the corresponding non‐chiral analogues of a C5‐methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC‐UV were investigated using four chiral stationary phases (CSPs: Lux Amylose‐2, Lux Cellulose‐1, Lux Cellulose‐2 and Lux Cellulose‐3). The best resolution was achieved using amylose tris(5‐chloro‐2‐methylphenylcarbamate) (Lux Amylose‐2), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X‐ray crystallographic analysis and comparative chiral HPLC‐UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC₅₀ values in the micromolar range. Chirality 25:400–408, 2013. © 2013 Wiley Periodicals, Inc.     

Effective resolution of racemic pirlindole at the preparative scale

Pirlindole, a racemic antidepressant drug, was recently resolved using the derivatization method coupled with preparative HPLC. In order to improve this technique, the use of amino acid derivatives as chiral derivatizing agents (CDA) was investigated. Among different residues, the (L)-phenylalanine methyl ester was found to be very effective to separate pirlindole enantiomers using a medium pressure liquid chromatographic (MPLC) method. This procedure is better adapted to preparative application than HPLC. Thus, several grams of the pirlindole antipodes were isolated and characterized. These two enantiomers permitted the study of the stereochemical influence at the pharmacological level. Chirality 11:261–266, 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.     

Synthesis of epimers of L-cyclopentenylglycine using enzymatic resolution

Both epimers of the naturally occurring nonproteinogenic amino acid L-cyclopentenylglycine, (2S,1'S)- and (2S, 1'R)-2-(cyclopent-2'-enyl)glycine, were obtained via a procedure involving condensation of 3-chlorocyclopentene with diethyl acetylaminomalonate, deethoxycarbonylation, chromatographic separation of the resulting two pairs of enantiomers, and enzymatic resolution of the racemates employing enantioselective hydrolysis of the ethyl ester group with alpha-chymotrypsin. The method was used for preparation of (13)C-labeled compounds of interest for biosynthetic tracer experiments. Enantiomeric purity of the products was determined by chiral HPLC on a Crownpak CR(+) column. The biologically active (2S,1'R) isomer was obtained as a pure compound and characterized for the first time. The (2R,1'R) and (2R,1'S) isomers were obtained as N-acetyl ethyl ester derivatives.     

Direct HPLC enantioseparation of chiral aptazepine derivatives on coated and immobilized polysaccharide-based chiral stationary phases

The direct HPLC enantioseparation of Mianserin and a series of aptazepine derivatives is accomplished on polysaccharide-based chiral stationary phases (CSPs). The resolutions are performed on the coated-type Chiralcel OD and Chiralpak AD CSPs and on the first commercially available immobilized-type Chiralpak IA CSP, in normal-phase and polar-organic modes. The complete separation of enantiomers of all racemates investigated was successfully achieved under at least one of CSP/eluent combinations employed. Pure alcohols such ethanol or 2-propanol, with a fixed percentage of DEA added, serve as valuable alternatives to the more common n-hexane-based normal-phase eluents in resolution of Mianserin on the AD CSP. In order to study the chiroptical properties of aptazepine derivatives, chromatographic resolutions are carried out at semipreparative scale using Chiralpak AD and Chiralpak IA as CSPs. Nonconventional dichloromethane-based eluents have permitted to expand the chiral resolving ability of the immobilized Chiralpak IA CSP and to perform mg-scale enantioseparations with an analytical-size column. Assignment of the absolute configuration of the separated enantiomers is empirically established by comparing their chiroptical data with those of structurally related Mianserin.