The importance of stereoselective determination of drugs in the clinical laboratory

About 56% of the drugs currently in use are chiral compounds, and 88% of these chiral synthetic drugs are used therapeutically as racemates. Only a few of these drugs qualify for a stereospecific determination in a clinical laboratory for therapeutic drug monitoring of patients. If the qualitative and quantitative pharmacokinetic and pharmacodynamic effects are similar, the enantiomers do not need to be separated. However, if the metabolism of the different stereoisomers is handled by different enzymes which are either polymorphic or can be induced or inhibited, and if their pharmacodynamic effects have differences either in strength or in quality, enantiospecific analysis is urgently needed. Unfortunately, there are many racemic drugs where the stereospecificity of the metabolism and/or the pharmacodynamic effects of the enantiomers is not known today. For these drugs, there is a great need for studies concentrating on these differences to improve treatment of the patients.     

Stereochemical facets of clinical β-blockers: An overview

The modern β-adrenergic agonists (β-blockers) possess one or more than one chiral center in their structure. Two enantiomers exhibit distinct pharmacodynamic and pharmacokinetic behaviors. Current progress in drug designing has resulted in the ability to understand the role of chirality in modern therapeutics. Furthermore, with a greater understanding of the molecular structure of precise drug targets, development of new drugs is directed towards the pure enantiomers instead of its racemates. The present review deals with a discussion on the stereochemical facets of chiral clinical β-blockers. This review provides details of stereo-selectivity in the pharmacological behavior of some of β-blockers and their metabolites. An effort has been made on highlighting the distinction between the therapeutic behavior of the racemic mixtures and pure enantiomers.     

Carbocyclic 3'-deoxyadenosine-based highly potent bisubstrate-analog inhibitor of basophilic protein kinases

Carbocyclic analogs of 3'-deoxyadenosine were synthesized as racemates and the resulting stereoisomers were separated by chromatography on a chiral column. The conjugation of obtained compounds with hexa-(D-arginine) via 6-aminohexanoic acid linker led to a highly potent inhibitor of several basophilic protein kinases with some selectivity towards cAMP-dependent protein kinase.     

Stereoselective determination of the epimer mixtures of itraconazole in human blood plasma using HPLC and fluorescence detection

Itraconazole is an antifungal drug widely used in a variety of fungal infections, which have become a significant public-health problem in recent decades. Itraconazole is a chiral drug consisting of two diastereoisomeric racemates, i.e., four stereoisomers. Data in the literature suggests that stereochemistry may play a significant role in the action and disposition of the drug and therefore stereoselective analytical methods for the determination of the drug in biological fluids are needed for the elucidation of that role. We report a stereoselective HPLC method that incorporates solvent extraction, the use of an internal standard, two chiral stationary phases in series, and fluorescence detection. The procedure is enantioselective and partially diastereoselective and provides the concentrations in blood plasma of the two epimer mixtures 2R,4S,2'R/2R,4S2'S and 2S,4R,2'R/2S,4R,2'S, respectively, each of which is a combination of the two epimers that differ in the configuration at the sec-butyl group. The analytical method has suitable sensitivity, recovery, precision, and accuracy. Analysis of the plasma of a human subject six hours after the oral administration of a single 200-mg dose of itraconazole showed a 3.4-fold difference between the concentrations of the epimer mixtures. The method has certain advantages over the published alternative procedure that uses LC-MS.     

Synthesis and absolute configuration of the stereoisomers of [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate,a muscarinic antagonist

The compound [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate 1 is a muscarinic antagonist characterized by the presence of three chiral atoms and eight possible stereoisomers. The binding affinities to the five cloned m₁–m₅ muscarinic receptors of the stereoisomers of 1 were previously investigated and proved to be related to the chirality of the molecules. The eight isomers are prepared through the synthesis of their racemates followed by chemical resolution as (+) and (−) tartrate or (+) and (−) dibenzoyltartrate salts. The isomers with cis-configuration of OH and COOH substituents of the cyclohexane are also obtained by coupling optically active (1S, 2S) or (1R,2R)-1-hydroxy-[1,1′-bicyclohexyl]-2-carboxylic acid with (S)- or (R)-1-diethylamino-2-propanol. Chiral GC and HPLC methods are used to determine their optical purity. The absolute configurations of the four cis- and four trans-isomers are established by stereospecific synthesis and X-ray crystallographic data. Chirality 9:713–721, 1997. © 1997 Wiley-Liss, Inc.     

High-performance liquid chromatographic separation of fluoroquinolone enantiomers: a review

Fluoroquinolones are antibacterial agents widely used clinically. In recent years, there has been an important development of new derivatives, and more than 7000 analogues have been described today. Different fluoroquinolones (FQ) have one or two chiral centers in their chemical structure and are available as racemates, diastereoisomers, or pure enantiomers. The clinical and pharmaceutical uses of these compounds need effective analytical procedures for quality control and pharmacodynamic and pharmacokinetic studies. This review article focuses on the high-performance liquid chromatographic separation of fluoroquinolone stereoisomers by the use of derivatization methods and ligand exchange (LE) or chiral liquid chromatography.     

Enantioselective and diastereoselective separation of synthetic pyrethroid insecticides on a novel chiral stationary phase by high-performance liquid chromatography

A novel chiral packing material for high-performance liquid chromatography (HPLC) was prepared by connecting (R)-1-phenyl-2-(4-methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to aminopropyl silica gel through 2-amino-3,5-dinitro-1-carboxamido-benzene unit. This chiral stationary phase was applied to the enantioselective and diastereoselective separation of five pyrethroid insecticides by HPLC under normal phase condition. To achieve satisfactory baseline separation an optimization of the variables of mobile phase composition was required. The two enantiomers of fenpropathrin and four stereoisomers of fenvalerate were baseline separated using hexane-1,2-dichloroethane-2-propanol as mobile phase. The results show that the enantioselectivity of CSP is better than Pirkle type 1-A column for these compounds. Only partial separations for the cypermethrin and cyfluthrin stereoisomers were observed. Seven peaks and eight peaks were observed for cypermethrin and cyfluthrin, respectively. The elution orders were assigned by using different stereoisomer-enriched products.     

Biological significance of the enantiomeric purity of drugs

The knowledge that enantiomers of chiral compounds may differ widely in biological activity, qualitatively as well as quantitatively, is not new. Nevertheless most of the pharmacological data available to date on chiral drugs are obtained from experiments with racemates which assume that the biological activity generally resides in one of the enantiomers. With the advancements made in stereospecific synthesis and stereoselective analysis of drugs pharmacologists are now offered new possibilities to explore the steric aspects of drug action. This survey will discuss pharmacological data obtained with enantiomer pairs of phenylethylamine derivatives which interact with adrenergic mechanisms. The degree of resolution is seldom specified in published work on stereoselectivity of drugs. In a recent study from our laboratory the enantiomers of the β₂-adrenoceptor agonist formoterol and their diastereomers have been evaluated. We found that the (R;R)-enantiomer was by far the most potent. However, the relative potencies obtained for the (R;S)-, (S;R), and (S;S)- isomers were critically dependent on the degree of enantiomeric purity. It is concluded that the certainty of potency ratios observed for chiral drugs is limited by the enantiomeric purity and by unspecific effects of the least active enantiomer at very high concentrations. © 1993 Wiley-Liss, Inc.     

Programmable enantioselective one-pot synthesis of molecules with eight stereocenters

We developed an enantioselectively catalyzed tandem synthesis of structurally and stereochemically complex molecules that forms four carbon-carbon bonds and sets eight stereocenters with high regio-, diastereo- and enantioselectivity. It can be programmed to yield different stereoisomers by varying only the order of combination of a common set of reagents and catalysts. We report what is to our knowledge the first synthesis of both enantiomers of a chiral compound using the same chiral catalyst.     

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.     

Enantioselective preparative HPLC separation of the HBCD-Stereoisomers from the technical product and their absolute structure elucidation using X-ray crystallography

1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a widely used flame retardant, which tends to persist in the environment and accumulates in biota. The six stereoisomers (three racemates named alpha-, beta-, and gamma-HBCD) of the technical mixture were isolated with high-performance liquid chromatography (HPLC). Direct separations were performed on a chiral stationary phase containing permethylated beta-cyclodextrin (NUCLEODEX beta-PM column) and the pure enantiomers of alpha-, beta-, and gamma-HBCD were physically characterized for the first time. The absolute configurations of all six isomers were determined by anomalous dispersion using single crystal X-ray crystallography. Optical rotations alphaD in tetrahydrofuran were +4.2/-4.0 (alpha-HBCD), +26.1/-27.5 (beta-HBCD), and +68.0/-66.3 (gamma-HBCD). The sense of rotation could be correlated with the absolute configurations of alpha-, beta-, and gamma-HBCD enantiomers and their order of elution on a chiral permethylated beta-cyclodextrin-bonded stationary phase. The diastereomersalpha-, beta-, and gamma-HBCD displayed distinctly different melting points as well as (1)H-, (13)C NMR, and IR spectra.     

Separation of enantiomers of drugs by capillary electrophoresis,part 7: Gamma-cyclodextrin as chiral solvating agent

Following an extended chiral drug screening program by capillary zone electrophoresis (CZE), the enantioseparation of 86 racemic drugs was tested with γ-cyclodextrin as a chiral solvating agent. Unified conditions were applied to all experiments. In total, 18 drug racemates were separated, 13 entries thereof that had not been separated at the lower CSA concentration applied in an earlier stage of the project. A comparison of the data with the results obtained for α- and β-cyclodextrin points to the significance of partial penetration (“side-on binding”) of aryl groups into the cyclodextrin cavity. Chirality 10:548–554, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis of optically active N-(2-pyridyloxiran-2-ylmethyl) benzenesulfonamide derivatives and their herbicidal activity

Novel herbicidally active sulfonamide compounds having a 2-arylsubstituted oxiranylmethyl structure are racemates due to a chiral carbon in the oxirane moiety. To clarify the stereochemical structure-activity relationship, we synthesized each enantiomer of 4-chloro-N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-3,N-dimethylbenzenesulfonamide and N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-N-methyl-5,6,7,8-tetrahydronaphthalene-2-sulfonamide by chemical methods including Sharpless asymmetric chlorohydroxylation. The results of herbicidal tests indicated that the (S)-isomers were the active forms.     

Synthesis of Optically Active N-(2-Pyridyloxiran-2-ylmethyl) benzenesulfonamide Derivatives and Their Herbicidal Activity

Novel herbicidally active sulfonamide compounds having a 2-arylsubstituted oxiranylmethyl structure are racemates due to a chiral carbon in the oxirane moiety. To clarify the stereochemical structure-activity relationship, we synthesized each enantiomer of 4-chloro-N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-3,N-dimethylbenzenesulfonamide and N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-N-methyl-5,6,7,8-tetrahydronaphthalene-2-sulfonamide by chemical methods including Sharpless asymmetric chlorohydroxylation. The results of herbicidal tests indicated that the (S)-isomers were the active forms.     

A consideration of the patentability of enantiomers in the pharmaceutical industry in the United States

During the last thirty years, concern over stereoselectivity of drug action has drawn a great deal of interest within the pharmaceutical field due to an improved understanding of the pharmacology and pharmacokinetics of enantiomers. Developing single enantiomers versus racemates or introducing a single enantiomer following the development of the racemic mixture appears to be the new trend. The intellectual property status of single enantiomers from racemates may be unclear. Drug discoverers and patent attorneys must examine the examples of the past to establish an appropriate pathway towards the development and intellectual property protection of chiral drugs. The review will focus on the patenting of an enantiomer in view of the prior art disclosure for the racemic mixture.     

Chiral liquid chromatography-tandem mass spectrometric methods for stereoisomeric pharmaceutical determinations

The characterization of the drug metabolism and pharmacokinetic (DMPK) profiles of stereoisomers is a fundamental aspect of the drug discovery and development processes. Therefore, chiral drug bioassays are very important to pharmaceutical and biomedical researchers. The recent developments in chiral liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry (LC-API-MS/MS) for the analysis of pharmaceuticals are reviewed. Various ionization techniques including electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric photoionization (APPI) interfaced with chiral liquid chromatographic methods are described in terms of their ionization efficiencies, matrix effects and limitations. Examples were selected to demonstrate the applicability of these methods for enantioselective bioanalysis.     

Effect of structural analogs of butaclamol (a new antipsychotic drug) on striatal homovanillic acid and adenyl cyclase of olfactory tubercle in rats.

The 3-isopropyl (I), 3-cyclohexyl (II) and 3-phenyl (III) analogs of the new antipsychotic drug butaclamol, which contains a 3-tertiary butyl group, and their respective (+)-enantiomers, but not (-)-enantiomers, caused a dose related elevation of rat striatal homovanillic acid concentration, indicative of an increased dopamine (DA) turnover; droperidol also exhibited this activity. The order of activity of the (+)-enantiomers was (butaclamol) approximately II greater than I greater than III. A decrease in striatal DA was observed with (+)-I and (+)-III at the highest dose used, but not at one-half the dose. Each analog antagonized the DA-induced increase in adenyl cyclase (EC 4.6.1.1) activity of olfactory tubercle homogenates, the order of activity of the racemates (except for II) AND (+)-ENANTIOMERS BEING (BUTACLAMOL) APPROXIMATELY I greater than III greater than II. The (+)-enantiomers of butaclamol and analogs were two to four times more potent than their respective racemates, with (+)-butaclamol and (+)-I displaying activity generally equivalent to fluphenazine. The respective (-)-enantiomers were ineffective indicating a stereochemical specificity for DA-receptor blockade. Such analogs presented should be of value in elucidating dopaminergic mechansims.     

Leucine-phenylalanine dipeptide-based N-mesyloxysuccinimides: synthesis of all four stereoisomers and their assay against serine proteases

The four stereoisomers of 3-(N-acetylleucylamino)-3-benzyl-1-[(methylsulfonyl)oxy]succinimid e have been prepared and shown to inhibit alpha-chymotrypsin and human neutrophil elastase. The structural and stereochemical features that affect the potency and selectivity of inhibition are discussed.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Synthesis and evaluation of two novel “mixed” chiral stationary phases deriving from tyrosine: Csps designed for the resolution of either π-acid or π-basic racemates

The synthesis and chromatographic evaluation of two novel chiral stationary phases (CSPs) deriving from (S)-tyrosine are reported. The chiral graft has been designed in order to bear both π-acid and π-basic sites, each one being connected to a distinct asymmetric centre. An intramolecular π-π interaction may take place within these CSPs, leading to an energetically favoured conformation of the chiral selector (CS). The enantiorecognition ability of these CSPs was investigated for various classes of either π-acid or π-basic racemates. It is shown that these CSPs are able to separate simultaneously π-acid and π-basic racemates. Finally, chiral recognition mechanisms and mobile phase optimization are discussed.