July 24-27, 2006, San Diego, CA Chiral Separations: A Tutorial Christine Aurigemma Pfizer Global...

July 24-27, 2006, San Diego, CA

Chiral Separations: A Tutorial

Christine AurigemmaPfizer Global Research & Development,

La Jolla, CAJuly 24, 2006


Outline

I. Stereochemistry Refreshera. Relationships of Stereoisomersb. Terminology

II. Chiral Separationsa. Why do we need chiral separations?b. Different approaches to enantiopure products

III. Chromatographic Chiral Separationsa. What is Chiral Recognition? 3-point ruleb. SFC vs. HPLCc. Types of CSP’sd. Screening optione. Problem solving

IV. Absolute Stereochemistry (Oliver McConnell)


Conformational isomers

Relationships of Stereoisomers

Courtesy of Brown/Foote, Organic Chemistry, 3/e, Figure 1Harcourt, Inc. items and derived items copyright 2002 by Harcourt, Inc. andhttp://www.chem.uic.edu/web1/OCOL-II/WIN/STEREO/ISOMER.HTM

Isomers: Compounds with the same molecular formula

Constitutional (or structural)isomers

Stereoisomers

Same atomconnectivity

Different atomconnectivity

Interconvert through rotation about asingle bond

Conformationalisomers or rotamers

Configurationalisomers

Not readily Interconvertible

EnantiomersDiastereomers

Chiral

w/ chiral centers (optically active)

w/ochiral centers (opt. inactive)

Geometric isomers

Achiral

Configurational isomers

mirror images Enantiomers

Cis, Trans (E,Z) isomers

cis and trans isomers

Constitutional (structural)isomers

mirror images at this carbonEnantiomeric

Not mirror imagesDiastereomers

Not mirror images at this carbonDiastereomeric


Chiral vs Achiral CompoundsAchiral Molecule: • Has no stereogenic center;

the carbon atom has less than 4 non-equivalent substituents attached

• has a plane of symmetry• one that is superimposable on

its mirror image (the two are identical)– i.e. nail, ball, a baseball bat

• Not optically active

Chiral Molecule: • Has one stereogenic center

(typically C, but can be N, P, etc.), which is attached to 4 different substituents asymmetric

• one that is not superimposable on its mirror image (the two are not identical)– i.e. hands, keys, shoes

• the two mirror image forms are called enantiomers

• Optically active

http://wps.prenhall.com/wps/media/objects/724/741576/Instructor_Resources/Chapter_05/Text_Images/FG05_01-10UN.JPG


Determination of Optical Activity

• Each enantiomer has an equal but opposite optical rotation; can be measured using optical rotation polarimeter

• One enantiomer rotates polarized light in a clockwise direction and is then designed as (+), or dextrorotatory

• The other enantiomer rotates polarized light in counter-clockwise direction and is the (-) enantiomer, or levorotatory

• Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they cancel each other out

Specific Rotation = []D =

l * c

where = observed rotation, l = cell length in dm, c = concentration in g/mL, and D is the 589nm light from a sodium lamp

©1999 William Reusch, All rights reserved (most recent revision 7/14/2006) [email protected]

mailto:[email protected]


Stereochemistry Terms

• Isomers: Compounds with the different chemical structures and the same molecular formula

• Stereoisomers: compounds made up of the same atoms but have different arrangement of atoms in space

• Enantiomers are the 2 mirror image forms of a chiral molecule– can contain any number of chiral centers, as long as each center is the

exact mirror image of the corresponding center in the other molecule– Identical physical and chemical properties, but may have different

biological profiles. Need chiral recognition to be separated.– Different optical rotations (One enantiomer is (+) or dextrorotatory

(clockwise), while the other is (-) or levorotatory (counter clockwise))• Racemate: a 1:1 mixture of enantiomers.

– Separation of enantiomers occurs when mixture is reacted with a chiral stationary phase to form 2 diastereomeric complexes that can be separated by chromatographic techniques

• Diastereomers: stereoisomers that are not enantiomers– Have different chemical and physical characteristics, and can be

separated by non-chiral methods.– Has at least 2 chiral centers; the number of potential diastereomers for

each chiral center is determined by the equation 2n, where n=the number of chiral centers


Outline



III. Chromatographic Chiral Separationsa. What is Chiral Recognition? 3-point ruleb. HPLC vs. SFCc. Types of CSP’sd. Screening optione. Problem solving


Racemate vs. Single Enantiomer

http://www.fda.gov/cder/guidance/stereo.htm;C&EN, May 5, 2003, pg. 56

• Single enantiomers of chiral active pharmaceutical ingredients (APIs) may have different:– Pharmacokinetic properties in animal models

• Absorption, distribution, metabolism and excretion– Pharmacological or toxicological effects

• Biologically “active” isomer may have desirable effects• Biologically “inactive” isomer may have undesirable side effects (i.e.

increased toxicity)

• Increased pressures by regulatory authorities to switch from racemic to single enantiomer APIs

• Development of chiral APIs raises issues regarding:– acceptable manufacturing control of synthesis and impurities– pharmacological and toxicological assessment of both

enantiomers– proper assessment of metabolism and distribution– proper clinical evaluation of these drugs


Chiral Blockbuster Drugs

Note: Sales figures from IMS HealthCourtesy of C&EN, September 5, 2005, Volume 83, Number 36, pp. 49-53

Nine of the top 10 drugs have chiral active ingredients

$53.5TOTAL

AntidepressantRacemateVenlafaxine3.7Effexor

AntiulcerantRacemateLansoprazole3.8Ogastro

Red blood cell stimulant

ProteinEpoetin alpha4.0Erypo

Anti-inflammatorySingle enantiomerFluticasone

BronchodilatorRacemate Salmeterol4.7Seretide/Advair

AntihypertensiveRacemateAmlodipine4.8Norvasc

AntipsychoticAchiralOlanzapine4.8Zyprexa

AntiulcerantSingle enantiomerEsomeprazole4.8Nexium

AntithromboticSingle enantiomerClopidogrel5.0Plavix

Cholesterol reducerSingle enantiomerSimvastatin5.9Zocor

Cholesterol reducerSingle enantiomerAtorvastatin$12.0 Lipitor

THERAPY CLASSFORM OF ACTIVE INGREDIENT(S)

ACTIVE INGREDIENT(S)

GLOBAL 2004 SALES ($ BILLIONS)

$53.5TOTAL

AntidepressantRacemateVenlafaxine3.7Effexor

AntiulcerantRacemateLansoprazole3.8Ogastro

Red blood cell stimulant

ProteinEpoetin alpha4.0Erypo

Anti-inflammatorySingle enantiomerFluticasone

BronchodilatorRacemate Salmeterol4.7Seretide/Advair

AntihypertensiveRacemateAmlodipine4.8Norvasc

AntipsychoticAchiralOlanzapine4.8Zyprexa

AntiulcerantSingle enantiomerEsomeprazole4.8Nexium

AntithromboticSingle enantiomerClopidogrel5.0Plavix

Cholesterol reducerSingle enantiomerSimvastatin5.9Zocor

Cholesterol reducerSingle enantiomerAtorvastatin$12.0 Lipitor

THERAPY CLASSFORM OF ACTIVE INGREDIENT(S)

ACTIVE INGREDIENT(S)

GLOBAL 2004 SALES ($ BILLIONS)


Examples

• Albuterol (anti-asthmatic inhalant)– D-albuterol may actually cause airway constriction– Levalbuterol (L-albuterol) avoids side effects

• Allegra (allergy medication)– Single enantiomer of Seldane that avoids life-

threatening heart disorders of Seldane

• Fluoxetine (generic name for Prozac, depression medication)– R-Fluoxetine – improved efficacy; minimizes side effects,

i.e. anxiety and sexual dysfunction. Other indications (eating disorders)

– S-Fluoxetine – use for treatment of migraines


• Chiral Synthetic Approach– Stereoselective or asymmetric syntheses– Biotransformation or Enzymatic resolution– Catalytic enantioselective processes

• Racemic Approach– Crystallization– Chiral salt resolution– CE (capillary electrophoresis)– SMB (simulated moving bed technology)– Chromatography (HPLC, SFC)

Approaches to Pure Enantiomers

Courtesy of Christina Kraml, Wyeth

Few SamplesLarge Scale

Many SamplesSmall Scale


Outline



III. Chromatographic Chiral Separationsa. What is Chiral Recognition? 3-point ruleb. HPLC vs. SFCc. Types of CSP’sd. Screening optione. Problem solving


• Chiral Recognition: Ability of chiral stationary phase, CSP, to interact differently with each enantiomer to form transient-diastereomeric complexes; requires a minimum of 3 interactions through:

– H-bonding– π-π interactions– Dipole stacking– Inclusion complexing– Steric bulk

• Five general types of CSPs used in chromatography:1. Polymer-based carbohydrates2. Pirkle or brush-type phases3. Cyclodextrins4. Chirobiotic phases 5. Protein-based

Chiral Chromatography

http://www.chemhelper.com/enantiomersep.html

CSP Biphenyl derivative


Classification of Chiral Stationary Phases (CSP)

1) Polymer-based Carbohydrates– Chiral polysaccharide derivatives, i.e. amylose and cellulose,

coated on a silica support– Enantiomers form H-bonds with carbamate links between

side chains and polysaccharide backbone– Steric restrictions at polysaccharide backbone may prevent

access of one of enantiomers to H-bonding site– Can be used with normal phase HPLC, SFC, RP-HPLC– Limitations: Not compatible with a wide range of solvents

other than alcohols

• Available columns:– i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-RH,

etc. from Chiral Technologies, Inc.– Chiralpak IA and IB…same chiral selectors as AD and OD, respectively, but

these are immobilized on the silica; more robust and has much greater solvent compatibilities

Courtesy of Chiral Technologies, Inc.


Naproxen examples using polymer-based CSPs

CH3

O

OH

MeO

Conditions:Chiralpak AD-HHexane/IPA/TFA, 80:20:0.1Flow: 1.0 mL/min

Conditions:Chiralpak AS-RHaq. H3PO4 (pH2)/ACN, 60:40Flow: 0.7mL/min

Conditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 80:20Flow: 5.0 mL/min

Conditions:Chiralpak AD-H, 100x4.6mmCO2/MeOH, 90/10Flow: 2.0 mL/min

Courtesy of Chiral Technologies, Inc.



2) Pirkle or Brush-type Phases: (Donor-Acceptor)– Small chiral molecules bonded to silica– More specific applications; strong 3-point interactions through 3 classes:

• π-donor phases• π-acceptor phases• Mixed donor-acceptor phases

– Binding sites are π-basic or π-acidic aromatic rings (π-π interactions), acidic and basic sites (H-bonding), and steric interaction

– Separation occurs through preferential binding of one enantiomer to CSP– Mostly used with normal phase HPLC, SFC. May get less resolution with RP-

HPLC; compatible with a broad range of solvents– Limitations: only works with aromatic compounds

• Available columns:• Whelk-O 1, Whelk-O 2, ULMO, DACH-DNB (mixed phases), -Burke 2, β-

Gem 1 (π-acceptor phases), Naphthylleucine (π-donor phases), from Regis Technologies, Inc.

• Phenomenex Chirex phases

Courtesy of Regis Technologies, Inc.


Naproxen examples using Pirkle-type CSP

(Normal phase)(Reversed phase)

Courtesy of Regis Technologies, Inc.



3) Cyclodextrin CSPs– Alpha, beta and gamma-cyclodextrins bond to silica and

form chiral cavities– 3-point interactions by:

• Opening of cyclodextrin cavity contains hydroxyls for H-bonding with polar groups of analyte

• Hydrophobic portion of analyte fits into non-polar cavity (inclusion complexes)

– One enantiomer will be able to better fit in the cavity than the other

– Used in RP-HPLC and polar organic mode– Limitations: analyte must have hydrophobic or aromatic

group to “fit” into cavity

• Available columns:– Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.– ORpak CDA (), ORpak CDB (), ORpak CDC () from JM Sciences

http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Brush


Conditions Results

Column: CYCLOBOND I 2000

Dimensions (mm): 250x4.6mm

Catalog Number: 20024

Mobile Phase: 10/90: CH3CN/1% TEAA, pH 4.1

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 23°C

Chart Speed (cm/min): 0.4cm/min.

Detection (nm): 254nm

Injection Volume (µL): 2.0µL

Sample Concentration (mg/mL): 5.0mg/mL

Peak1 16.1 Peak2 18.1

Chlorpheniramine example using

Cyclodextrin-type CSP

http://www.astecusa.com/applications/result_Mod.asp

chlorpheniramine



4) Chirobiotic Phases– Macrocyclic glycopeptides linked to silica – Contain a large number of chiral centers

together with cavities for analytes to enter and interact

– Potential interactions:• π-π complexes, H-bonding, ionic interactions• Inclusion complexation, steric interactions

– Capable of running in RP-HPLC, normal phase, polar organic, and polar ionic modes

• Available columns:– Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2

(Teicoplanin), Chirobiotic R (Ristocetin A) from Astec

http://www.raell.demon.co.uk/chem/CHIbook/chiral.htm#Macrocyclic


Peak1 8.78 Peak2 10.48

Conditions Results

Column: CHIROBIOTIC V

Dimensions (mm): 250x4.6

Catalog Number: 11024

Mobile Phase: 10/90:THF/0.1% TEAA, pH7

Flow Rate (mL/min): 1.0 mL/min.

Temp (oC): 25°C

Chart Speed (cm/min): 0.5

Detection (nm): 254

Injection Volume (µL): 2

Sample Concentration (mg/mL): 5

Naproxen example using Chirobiotic-type CSP

http://www.astecusa.com/applications/result_Mod.asp

Naproxen



5) Protein-based CSPs– Natural proteins bonded to a silica matrix– Proteins contain large numbers of chiral centers and interact

strongly with small chiral analytes through:• Hydrophobic and electrostatic interactions, H-bonding

– Limitations: • Requires aqueous based conditions in RP-HPLC • Analyte must have ionizable groups such as amine or acid. • Not suited for preparative applications due to low sample

capacity

• Available columns:– Chiral AGP (-glycoprotein) from ChromTech– HSA (human serum albumin) from ChromTech– BSA (bovine serum albumin) from Regis Technologies


Naproxen examples using Protein-based type CSP

http://www.chromtech.se/nap-2x.htm

Human Serum Albumin CSP Acid glycoprotein CSP


Selecting a CSP

• General use column with no solubility issues

Polymer-based phases • Specific applications; solubility issues

Pirkle-typeChirobiotic phases

• SFC onlyPolymer-based, Pirkle-type, Chirobiotic

• Biological SamplesProtein-based phases


Suggested Applications of CSPs

ChirobioticPeptides

PirkleOxazolindones

Pirkle, cellulose, proteinNSAIDS

Cellulose, PirkleDihydropyridines

Cellulose, PirkleAlkaloids

Pirkle, celluloseCyclic ketones

Cellulose, PirkleLactones

Protein, PirkleAromatic drugs

ProteinCyclic drugs

CyclodextrinsPolycyclic aromatic

hydrocarbons

Pirkle, celluloseβ-Lactams

PirkleBinaphtols

Pirkle, ChirobioticHydantoins

PirkleSuccinamides

Pirkle, ChirobioticAmino alcohols

PirkleThiols

CyclodextrinsMetallocenes

CyclodextrinsCrown ethers

PirkleUreas

PirkleCarbamates

Pirkle, cellulose, protein, ChirobioticSulfoxides

Pirkle, cellulose, ChirobioticEsters

Protein, cellulose/amylose, Pirkle, cyclodextrins, ChirobioticAmides

Protein, cellulose/amylose, Pirkle, cyclodextrinsAlcohols

Protein, cellulose/amylose, Pirkle, cyclodextrins, ChirobioticAmines

Cyclodextrins, protein, ChirobioticAmino Acids

Protein, cellulose/amylose, Pirkle, ChirobioticAcids

Type(s) of CSPClass of Compound

ChirobioticPeptides

PirkleOxazolindones

Pirkle, cellulose, proteinNSAIDS

Cellulose, PirkleDihydropyridines

Cellulose, PirkleAlkaloids

Pirkle, celluloseCyclic ketones

Cellulose, PirkleLactones

Protein, PirkleAromatic drugs

ProteinCyclic drugs

CyclodextrinsPolycyclic aromatic

hydrocarbons

Pirkle, celluloseβ-Lactams

PirkleBinaphtols

Pirkle, ChirobioticHydantoins

PirkleSuccinamides

Pirkle, ChirobioticAmino alcohols

PirkleThiols

CyclodextrinsMetallocenes

CyclodextrinsCrown ethers

PirkleUreas

PirkleCarbamates

Pirkle, cellulose, protein, ChirobioticSulfoxides

Pirkle, cellulose, ChirobioticEsters

Protein, cellulose/amylose, Pirkle, cyclodextrins, ChirobioticAmides

Protein, cellulose/amylose, Pirkle, cyclodextrinsAlcohols

Protein, cellulose/amylose, Pirkle, cyclodextrins, ChirobioticAmines

Cyclodextrins, protein, ChirobioticAmino Acids

Protein, cellulose/amylose, Pirkle, ChirobioticAcids

Type(s) of CSPClass of Compound

Compiled from Snyder, et. al, “Practical HPLC Method Development”, 2nd ed., John Wiley and Sons, Inc. 1997, p. 549


Chiral SFC vs. HPLC

• Advantages– Reduced solvent

• Amounts (CO2 reduces liquid waste)– Reduced toxicity

• Solvent types (alkanes, chlorinated, etc)• CO2 has a net zero environmental impact

– Safety• Reduce flammability

– Separation speed/efficiency

• Disadvantages– Equipment costs– Maintenance/robustness– Solubility


= 1.76Run time = 20.5 minutesFlow rate = 1.5 mL/min

= 1.35Run time = 10 minutesFlow rate = 0.4 mL/min

HPLC (normal phase) SFC (normal phase)

Flurbiprofen examples using HPLC and SFC

http://www.registech.com/chiral/sfcappguide2006.pdf


Chiral Screen

• Mobile phases: CO2 + methanol or isopropanol• Columns:

– Chiralpak AD-H, AS-H– Chiralcel OD-H, OJ-H– Chiralpak IA (immobilized AD)

Column 1

Column 2

Column 3

Column 4

Column 5

DetectorSFC

Solvent selectorvalve Column selector

valve

Solvents


Daicel Chiralcel OD-H25% MeOH, 140 bar

Daicel Chiralpak AD-H30% MeOH, 140 bar

> LOADABILITY

Changing Stationary Phase


Problem Solving Approaches • Derivatization of final products and

intermediates– Use of protecting groups such as t-BOC and CBZ (carbobenzyloxy)– CBZ derivatization of chiral primary and secondary amines

(common intermediates or final products of enantioselective synthesis)

– Adding CBZ can improve compound solubility, enables high efficiency purifications through repetitive, stacked injections

– Enhances chiral recognition and improves 3-point interactions; improves baseline separation ability by either HPLC or SFC

– CBZ protecting group easily attached and removed during synthetic processes

Kraml, Christina et. al ,“Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatoGraphy”, J. of Chrom A, 1100 (2005) 108-115..

• Acylation of amine with benzyl chloroformate• Amine is regenerated by catalytic hydrogenolysis using palladium on carbon• Product is isolated by simple filtration and evaporation of the solvent

N

OO Pd/C

H2

PhCH2OCOCl

iPr2NEt

NH

NH

+ CO2 + toluene


CBZ-Derivatization

• 47.5 g per day

• Isolated 70 g

• 35.4 hrs. purification time

underivatized Purify: ~2g/hr

min0 0.5 1 1.5 2 2.5 3 3.5

mAU

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min0 0.5 1 1.5 2 2.5 3 3.5

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Aurigemma, C., BSAT 2005, Boston, MA


• Addition of strong acid additives to mobile phase – Especially useful for separation of chiral amines – 0.1% ethanesulfonic acid (ESA) added to ethanol, or

0.1% methanesulfonic acid (MSA) added to methanol will cause formation of ion pairs with the amine to increase chances of successful enantioseparation

Courtesy of Roger Stringham, Chiral Technologies, Inc


•Use of Basic Additives to Mobile Phase Use of Basic Additives to Mobile Phase andand Sample solventSample solvent

*Trans()-2-Phenylcyclopropanamine•HCl, CAS No. 1986-47-6Aurigemma, C., BSAT 2005, Boston, MA

Analytical SFC

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(S,S) Whelk-O 1, 250x4.6mm, 10u i.d. (Regis Technologies, Inc.) 40% IPA w/ 0.1% IPAm 2.5 mL/min @ 140 bar

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No additive in sample solvent

Isopropylamine inMobile phase only

95908580757065605550454035302520151050

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Result: better peak shapes, allowing for high throughput purificationsthrough stacked injections and yielding pure enantiomers

Preparative


NO residual base in collected sample

No IPAm in sample solvent

IPAm added to sample solvent

IPAm

•Use of Basic Additives to Sample Solvent onlyUse of Basic Additives to Sample Solvent only

Aurigemma, C., BSAT 2005, Boston, MA


Summary

• Direct separations of enantiomers achieved by changing CSP’s

• Solubility issues can be resolved by adding CBZ or another protecting group

• Poor peak shapes can be overcome by addition of additives to MP, MP + sample solvent, or sample solvent only


Questions??

July 24-27, 2006, San Diego, CA Chiral Separations: A Tutorial Christine Aurigemma Pfizer Global...

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Transcript of July 24-27, 2006, San Diego, CA Chiral Separations: A Tutorial Christine Aurigemma Pfizer Global...