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Technical Guide ToChiral HPLCSeparationsReview ofChiral PhasesandMethod Development Strategies SM

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Technical Guide To Chiral HPLC SeparationsColumn Selection and Method Development TechniquesIntroduction and ObjectivesEffective separations of enantiomers have become increasingly important to thepharmaceutical and agrochemical industry, as well as to biotechnology and mostother areas of natural products chemistry. The rapid introduction of optically active medicinal drugs and pesticides, along with increasing government regulation,necessitates that rapid, sensitive and reliable stereochemical methods be devisedfor their analysis. Chromatography is by far the most powerful and sensitive analytical technique for resolving enantiomers, and chiral HPLC is probably the mostversatile and important tool employed today.This guidebook introduces the concepts of chirality and chiral separations by HPLC,and explores in detail the approach to column selection and method development.It summarizes the techniques used to develop and optimize chiral separations, andteaches by example and illustration. Data have been extracted from a large numberof applications developed by both Phenomenex and our customers.A systematized approach to column selection is presented, and is highly dependent on the structure of your compound. Ultimately, however, column selectionbecomes an empirical process, based on what has already been shown to work,or by exploring column utility for related compounds, or more simply, by trial anderror. Once one or more columns are screened for their utility, optimization of theseparation on a given column is a more methodical and straightforward process.In this guidebook our intention is to suggest by illustration general separationstrategies you can use to successfully resolve and purify enantiomers directlyand without difficulty. There are few hard and fast rules to follow. But in view ofthe applicability and versatility of the chiral stationary phases described here, youshould have confidence that you are working with the most sophisticated toolsavailable today for chiral HPLC.Phenomenex maintains a dedicated chiral separation services laboratory whichcan assist you in developing or improving your chiral separation. Please call on usfor help. We can assist you in column selection, method development, scale up orany other aspect of chiral chromatography. Phenomenex is the leader in advancedchiral separations.

Guidebook OutlineChiral HPLC Separations of Racemic Compounds &Chirality&The Chirotechnology Revolution&HPLC Separation Techniques&Pirkle-Concept Separations&Method Development Techniques&Pharmaceutical Applications

Chirality and Molecular StructureCauses of Molecular Asymmetry due to the arrangement of atoms in spaceChiral CenterC, N, P, S or BChiral AxisAllene substitutionChiral PlaneAtropisomerismMolecular StrainHelicenes,Natural polymersSubstituted BiarylsOrtho-bridging ofBiaryls

Chiral Drugs and their Applicationsas Single Isomers or Racemic Mixturesnaturalsemisynthetic475non-chiral6sold as single isomer461chiral469sold as racemate8Drugs1675non-chiral720sold as single isomer58synthetic1200chiral480sold as racemate422 40% of all man-made synthetic drugs are chiral 60% of all pharmaceuticals are chiral 45% of all chiral drugs are sold as racemates

Stereospecific ActivityS - ( ) - KETOPROFEN( a n a l g e s i c , a n t i i n fl a m m a t o r y )R - (-) - KETOPROFEN(slows periodontalbone loss)2S.3R ( - )levopropoxypheneantitussive2R.3S ( )dextropropoxypheneanalgesicDARVON NOVRADEnantiomers with different types of action. The analgesic action is incompatible with theantitussive, respiration depressant action. Both enantiomers are marketed, one as an analgesic, the other as an antitussive (anti-cough) drug. Their chemical mirror-type relationshipis reflected in the names Darvon and Novrad [9].

Enantiomers May Confer Benefits OverRacemates for Therapeutic UsesProperties of racematePotential benefits of enantiomerOne enantiomer is exclusively activeReduced dose and load on metabolismThe other enantiomer is toxicIncreased latitude in dose and broaderuse of the drugEnantiomers have different pharmacokineticsBetter control of kinetics and doseEnantiomers metabolized at differentrates in the same personWider latitude in setting the dose;reduction in variability of patients’responsesEnantiomers metabolized at differentrates in the populationReduction in variability of patients’responses; greater confidence in settinga single doseOne enantiomer prone to interactionwith key detoxification pathwaysReduced interactions with other common drugsOne enantiomer is agonist, the otherantagonistEnhanced activity and reduction of doseEnantiomers vary in spectra of pharmacological action and tissue specificitySource: Chiros Increased specificity and reduced sideeffects for one enantiomer; use of otherenantiomer for different indication

These drugs are Best Candidatesfor Racemic SwitchesCardiovascularAnti-inflammatory and analgesicsAcebutolol AlprenololCicloprofen CorticosteroidsAtenolol BetaxololDihydroxythebaine FenbuphenBisprolol BopindololFenoprofen FlurbiprofenBucumolol BufetololIbuprofen IndoprofenBufuralol BunitrololKetoprofen MinoxiprofenBupranolol ButofilololPirprofen SteroidsCarazolol CarvedilolSuprofen TriamcinoloneCurteolol DisopyramideDobutamine IndenololMepindolol MetipranololAnticancerCytarabineMetoprolol NadololNicardipine OxpranololPindolol PropanololAntibiotics, antiinfectives,antiviralsSotalol ToliprololCiprofloxacinVerapamil XibenololOfloxacinCentral nervous systemNorfloxacinHormones, genitourinaryDobutamine FluoxetineBenzyl glutamateButoconazoleKetamine LorazepamCalcitoninEstradiolMeclizine olamine estin nitanolTestosteronePolycloramphetamine TomoxetineToloxaton ViloxazinAntihistamine and l/salbutamol MetaproteranolTerbutalineSource: Technology Catalysts InterationalA Racemic Switch is the redevelopment of a drug now marketed as a racemate to the pure enantiomer.

Isomer Separation Flow ChartISOMERSCONSTITUTIONALSTEREOISOMERS ALLY ACTIVESEPARATION OFDIASTEREOMERSBY HPLCNON-TRANSIENTCOMPLEX USINGCHIRAL DERIV.AGENTS (CDA)ACHIRALCHROMATOGRAPHYSILICA, ALUMINA, ETC.TRANSIENT COMPLEX(WEAKLY ASSOCIATED)CHIRAL MOBILEPHASE ADDITIVE(CMPA)CHIRAL STATIONARYPHASE (CSP)CLASSIFICATIONTYPES I - V

Classification ofChiral Stationary Phases us chiral selectorsAttractive interactions(Pirkle)Ionic or covalent bonding Hydrogen BondingCharge transfer (π-π interaction)Dipole stackingIIHELICAL POLYMERSCellulose derivativesPoly methacrylatesInsertion complexesAttractive interactivesPoly acrylamidesIIICAVITYCyclodextrinsInclusion complexesCrown ethersIVLIGAND EXCHANGEAmino acid-metal complexDiastereomeric metal complexVPROTEINa-acid glycoproteinHydrophobic interactionsBovine Serum AlbuminPolar interactionsMACROCYCLICAntibioticVIAttractive interactionsGLYCOPEPTIDEHydrogen BondingInclusion complexesPolar interactionsCharge transfer (π-π interaction)

Pirkle-Concept CSPsMechanism of InteractionEnantiorecognition involves hydrogen bonding, dipolar and charge transfer interactions and steric hindranceachiralspacerchiralmoietytype of interactions:dipole / dipolep/psilicahydrogen bondingsteric hindranceClass-I CSPs and types of interactions that can be involved during the chiral recognition process.soluteenantiomerschiralselectorThe three-point interaction model advanced by Dalgliesh10

Chiral Stationary Phases (CSPs)CSP TYPE II. INSERTION COMPLEXHELICAL POLYMERS (Natural CELLULOSES andDERIVATIVES and VARIOUS SYNTHETIC sanAcetatesDextranEsterslinear poly[1 - 4 - b - D -glucose]CarbamatesSynthetic:(a) Use of chiral substituent:polymerizationcrosslinkingchiral monomerchiralpolymer network(b) Use of chiral catalyst:PolymethacrylatesPolyacrylamidesisotactic (stereoregular)polymerAdopts a single helicalconformation11

Chiral Stationary Phases (CSPs)CSP TYPE III. INCLUSION (CAVITY) COMPLEXCYCLODEXTRINS and yclodextrinb-Cyclodextring-CyclodextrinThe structure of a, b and g cyclodextrin.Schematic showing the reversible inclusion complex formation typical of cyclodextrin bonded stationary phase.CSP Type IV. Ligand ExchangeDiastereomeric Metal ComplexIllustration of the principle of chiral ligand exchange used for chromatographic optical resolution.Reversible formation of diastereomeric metal complexes is obtained in a reaction between achiral selector (left-hand side) and the respective enantiomers (right-hand side).12

Chiral Stationary Phases (CSPs)CSP Type V. ProteinAGP, BSA, HSA, OVOMUCOIDCSP Type VIMacrocyclic/antibioticvancomycin, teicoplanin, ristocetin13

The Chirex Advantage14FWide variety of supportsFHigh enantioselectivityFHigh efficiency, covalently bound columnsFChemically stable and rigid supportsFChoice of elution order (opposite CSPs available)FPacked columns from minibore to preparative

Chiral Stationary PhasesStationary phases in Chirex columns contain various chiral components. They are classified intothree types: amide, urea and ligand exchange types. In the amide type, asymmetric carbon atomsare bonded directly with the CONH group, and in the urea type, asymmetric carbon atoms with theNHCONH group, and they are chemically (covalently or ionically) bonded to silica gel. In the ligandexchange type, the chiral components are coated hydrophobically on ODS.CHIREXChiral ters, amides, carboxylic3005(R)-I-naphthylglycineRPacids, alcohols, ketones3010(S)-valineRPcarboxylic acids (including3011(S)-tert-leucineRPZ-amino acids, BOC-aminoRPacids, benzoylamino acids)Amide typeUrea phthyl)ethylamineNP(S)-proline,amines, amides, aminoNP3017(S)-I-(a-naphthyl)ethylaminealcohols, alcohols, esters,(S)-prolinephosphoric ne-2-carboxylic nge type3126(D)-penicillamineRPa-amino acids, a-hydroxy acids*NP : Normal phase modeRP : Reverse phase mode, typically consisting of ammonium acetate in methanolEnantiomeric stationary phases are available except for 3022, and on this phase the elution order of enantioners areinverted.15