Application of preferential crystallization for racemic compound integrating thermodynamics, kinetics and optimization 1

VIII List of figures...X Nomenclature ...XV Chapter 1 Introduction...1 Chapter 2 Literature review ...8 2.1 Enzymatic separation ...10 2.2 Separation by chiral chromatography ...11 2.3 C

APPLICATION OF PREFERENTIAL CRYSTALLIZATION FOR RACEMIC COMPOUND INTEGRATING THERMODYNAMICS, KINETICS AND

OPTIMIZATION

LU YINGHONG

NATIONAL UNIVERSITY OF SINGAPORE

2008

Application of Preferential Crystallization for Racemic Compound Integrating Thermodynamics, Kinetics and

Optimization

LU YINGHONG

(B Eng., Sichuan University

M Eng., Xian Jiaotong University)

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2008

Lots of thanks go to Prof Zeng Huachun for his kind assistance

Special thanks go to Dr Wang Xiujuan for her valuable scientific discussion and many critical comments on my research work

It is also my pleasure to express my gratitude to all the staff and students in Prof Ching’s group for their friendship, helps and encouragement

In addition, I would acknowledge National University of Singapore for providing me this opportunity to pursue my PhD degree and the research scholarship

At last, I wish to thank my husband, my parents and my little baby girl for their consideration, help and encouragement, which are huge support behind this work

II

Table of contents

Acknowledgment I Table of contents II Summary VI List of tables VIII List of figures X Nomenclature XV

Chapter 1 Introduction 1

Chapter 2 Literature review 8

2.1 Enzymatic separation 10

2.2 Separation by chiral chromatography 11

2.3 Chiral crystallization 14

2.3.1 Characterization of the racemic species 15

2.3.2 Resolution by crystallization of diastereoisomers 18

2.3.3 Optical resolution by direct crystallization 20

2.3.3.1 Separation of enantiomers by direct crystallization of their racemate 20

2.3.3.2 Preferential crystallization of enantiomeric enrichment of racemic compound 24

2.3.4 Process of preferential crystallization 28

2.4 Objective of the present research work 30

Chapter 3 Characterization of two kinds of racemic compounds: Mandelic acid and Ketoprofen 37

3.1 Introduction 38

III

3.2 Materials and methods 39

3.2.1 Materials 39

3.2.2 Fourier Transform Infrared Spectroscopy (FTIR) 40

3.2.3 Raman spectroscopy 40

3.2.4 Powder X-ray Diffraction (PXRD) 40

3.2.5 Differential Scanning Calorimetry (DSC) 41

3.3 Results and Discussion 41

3.3.1 Characterization by analytical spectroscopic techniques 41

3.3.1.1 Fourier Transform Infrared Spectra 42

3.3.1.2 Raman spectroscopy 44

3.3.1.3 Powder X-ray Diffraction 45

3.3.2 Characterization by thermal analysis and binary phase diagram 47

3.3.2.1 Binary phase diagram of mandelic acid 47

3.3.2.2 Binary phase diagram of Ketoprofen 54

3.4 Conclusion 59

Chapter 4 Crystallization thermodynamics: solubility and metastable zone width of mandelic acid and ketoprofen 64

4.1 Introduction 62

4.2 Method and Experiments 65

4.2.1 Apparatus 65

4.2.2 Solubility and metastable zone width (MSZW) 66

4.3 Results and discussion 68

4.3.1 Solubility and metastable zone width for the mandelic acid 68

4.3.1.1 Solubility and ternary phase diagram for mandelic acid 68

4.3.1.2 Metastable zone width for the mandelic acid 71

4.3.2 Solubility and metastable zone width for ketoprofen 76

4.3.2.1 Solubility and ternary phase diagram for ketoprofen 76

4.3.2.2 Metastable zone width for ketoprofen 80

4.3.2.3 Fractional Experiment Design 81

4.3.2.4 Analysis of the effects influencing metastable zone width of ketoprofen 84

IV

4.4 Conclusion 91

Chapter 5 Crystallization kinetics of mandelic acid and ketoprofen 93

5.1 Introduction 94

5.2 Mathematical model 97

5.2.1 Method of moment analysis 97

5.2.2 Method of Laplace transformation 99

5.3 Experimental Procedure 100

5.4 Results and discussion 101

5.4.1 Crystal nucleation and growth kinetics for the (S)-MA and (RS)-MA 101

5.4.1.1 Crystal suspension density and supersaturation 101

5.4.1.2 Crystal size distribution (CSD) 104

5.4.1.3 Kinetic evaluation on the measured data 106

5.4.2 Crystal nucleation and growth kinetics for the (S)-Kp and (RS)- Kp 110

5.4.2.1 Solubility 110

5.4.2.2 Crystal size distribution 112

5.4.2.3 Crystal growth and nucleation kinetics evaluation 114

5.5 Conclusion 118

Chapter 6 Systematic preferential crystallization process of mandelic acid 120

6.1 Introduction 121

6.2 Experiment 123

6.2.1 Semi-preparative HPLC separation of mandelic acid 123

6.2.2 Direct crystallization operation 123

6.3 Results and discussion 124

6.3.1 Semi-preparative HPLC separation of mandelic acid 124

6.3.2 Preferential crystallization operation for mandelic acid 127

6.3.2.1 The progression of preferential crystallization 127

6.3.2.2 Optimal operation profile 132

6.3.2.3 Optical purity and crystal size distribution 137

6.3.2.4 Seed size effect on crystal size distribution 143

V

6.4 Conclusions 145

Chapter 7 Application of direct crystallization for racemic compound ketoprofen 147

7.1 Introduction 148

7.2 Experiment and methods 150

7.2.1 HPLC collection of ketoprofen 150

7.2.2 Direct crystallization process 150

7.3 Result and discussion 151

7.3.1 Semi-preparative HPLC separation of Ketoprofen 151

7.3.2 Preferential crystallization operation for ketoprofen 153

7.4 Conclusion 161

Chapter 8 Conclusions and Future work 162

8.1 Conclusions 163

8.2 Future work 166

References 168

List of publications 193

VI

Summary

In comparison to other chiral separation methods, the preferential crystallization

is one of the simplest and most efficient processes Typically, it was used for the enantioseparation of a racemic conglomerate However, the applicability of preferential crystallization to racemic compounds would significantly widen the potential of crystallization based techniques for enantioseparation because racemic compounds occupy 90-95% of all racemates For a racemic compound system, it is crucial to keep the freedom of supersaturation of the racemate in its metastable zone

to avoid its spontaneous nucleation and control the supersaturation of the target enantiomer as the spontaneous nucleation of the target enantiomer may easily initiate the spontaneous nucleation of its racemate In our group’s previous work (Wang and Ching, 2006), the concept of critical supersaturation control for preferential crystallization was introduced and the strategy was applied to racemic conglomerate Therefore, in this dissertation, the objectives are to extend and modify this strategy to two kinds of racemic compound systems: mandelic acid and ketoprofen A systematic preferential crystallization was studied on solubility, metastable zone, kinetics and supersaturation control profile to obtain crystal product with good quality

Two typical types of racemic compounds, namely favorable racemic compound (mandelic acid) and unfavorable racemic compound (ketoprofen), were characterized

by various spectroscopic techniques, thermal analysis and phase diagrams construction The solubilities and metastable zone widths (MSZWs) were studied for both systems The MSZWs of racemate are different from those of pure enantiomer for a racemic compound system The MSZWs of mandelic acid were favorable for

VII

preferential crystallization process, while the MSZWs of high mole percent (S)-

ketoprofen were narrow, which indicated it was hard to control the supsaturation level

of pure enantiomer to inhibit its spontaneous nucleation to induce nucleation of racemate during the crystallization process The classical Laplace transform analysis and moment analysis were used for deriving the crystal growth rate and nucleation rate in the batch crystallization process for enantiomer and racemate of mandelic acid and ketoprofen The enantiomer and racemate show different characteristics in crystal nucleation and growth

Through the study on the direct crystallization progression for the mandelic acid and ketoprofen system, it was found that the optically pure product could be obtained by direct crystallization with seeding within certain safe supersaturation limit for a racemic compound So, it is important to control supersaturation degree in preferential crystallization Based on the thermodynamic and kinetic consideration, an optimal temperature control profile was derived to control the critical supersaturation

in order to inhibit the induced nucleation of the racemate for the mandelic acid Compared with the forced and linear cooling profile, the final crystal products of the proposed control cooling profile were almost optically pure with high yield and good crystal size distribution The results suggest that it is crucial and helpful to combine thermodynamics and kinetics to establish the control strategy This is especially critical for the unfavorable ketoprofen system due to its high eutectic composition and narrow metastable zone widths, which cause narrow feasible region and more difficulty to control for direct crystallization Direct crystallization alone could be less effective and less economical as an enantioseparation process for the ketoprofen system

VIII

List of tables

Table 2.1 Resolution methods 9

Table 3.1 Thermodynamic properties of (S)- and (RS)- MA 51

Table 3.2 The melting temperature and eutectic temperature of different mole fraction of (S)-MA 53

Table 3.3 Thermodynamic properties of (S)- and (RS)- Kp 57

Table 3.4 The melting temperature and eutectic temperature of different mole fraction of (S)-Kp 58

Table 4.1 Solubility data (g/ml) for different mole percent of (S)-MA 69

Table 4.2 Solubility data for different mole percent of (S)-Kp 78

Table 4.3 Levels for each of four factors 83

Table 4.4 The design of L9 with coded levels of factor 83

Table 4.5 The solubility data of (RS)-Kp, 0.94 mole fraction of (S)-Kp, and (S)-Kp (mg/ml) 84

Table 4.6 Worksheet for a L9 design for metastable zone widths 85

Table 4.7 Calculation average responses for three-level experiment for (RS)-Kp 86

Table 4.8 Calculation average responses for three-level experiment for 0.94 mole fraction of (S)-Kp and (S)-Kp 87

Table 5.1 Crystallization kinetics measurement of (S)-MA 102

Table 5.2 Crystallization kinetics measurement of (RS)-MA 102

Table 5.3 Estimated (S)-MA crystal nucleation rate B and growth rate G with s plane analysis from the experiment 106

Table 5.4 Estimated (RS)-MA crystal nucleation rate B and growth rate G with s plane analysis from the experiment 107

Table 5.5 Solubility of (RS)-Kp and (S)-Kp 111

Table 5.6 The estimated linear growth rate and nucleation rate for (RS)-Kp 114

IX

Table 5.7 The estimated linear growth rate and nucleation rate for (S)-Kp 116

Table 6.1 The optical purity of the final crystal products with different

cooling degree for mandelic acid 130Table 6.2 The optical purity of final crystal product 139 Table 7.1 The optical purity of the final crystal products with different

cooling degree for ketoprofen 159

X

List of figures

Fig 1.1 A pair of chiral enantiomorphic forms .2

Fig 2.1 Homochiral and heterochiral packing alternative 16

Fig 2.2 Typical phase diagram of various racemic species 17

Fig 2.3 A solubility isotherm ternary phase diagram for a conglomerate; (a) the composition of the solution changes during the course of the crystallization; (b) Preferential crystallization procedure 23

Fig 2.4 Binary phase diagram for a racemic compound .25

Fig 2.5 Ternary phase diagram for a racemic compound .27

Fig 2.6 Ternary phase diagrams for a racemic compound: (a) unfavorable; (b) more favorable; (c) most favourable .32

Fig 2.7 Chemical structure of (R)- and (S)- mandelic acid .32

Fig 2.8 Chemical structure of (R)- and (S)- ketoprofen 33

Fig 3.1 The FTIR patterns of the pure (S)-MA and (RS)-MA .43

Fig 3.2 Infrared spectra of (S)-Kp and (RS)-Kp 43

Fig 3.3 The Raman curve of pure (S)-MA and (RS)-MA .44

Fig 3.4 The Raman curve of pure (S)-Kp and (RS)-Kp .45

Fig 3.5 The PXRD patterns of pure (S)-MA and (RS)-MA .46

Fig 3.6 PXRD patterns of (RS)-Kp and (S)-Kp 47

Fig 3.7 Typical DSC curve of different mole percent of (S)-MA 49

Fig 3.8 The binary phase diagram of MA 54

Fig 3.9 DSC curves of different mole percent (S)-Kp .56

Fig 3.10 The binary phase diagram of Kp .59

Fig 4.1 Typical super solubility diagram Line AC: Cooling a saturated

solution; Line AC': Concentrating a saturated solution by

XI

evaporation of solvent; Line AC'': A combination of cooling and

evaporation 63

Fig 4.2 Experimental set-up: 1– Computer; 2- LabMax; 3- Julabo cooling/heating refrigerator; 4- Heavy duty stirrer; 5- Condenser; 6- Temperature sensor; 7- 1 liter double-wall crystallizer; 8- FBRM; 9- Balance; 10- Solvent bottle; 11- Dosing pump .66

Fig 4.3 Solubility of MA with different mole percent in water .70

Fig 4.4 The ternary phase diagram of MA in Water 70

Fig 4.5 Experimental MSZWs of S-MA in water at different cooling rates 72

Fig 4.6 Experimental MSZWs of 80% mole percent of (S)-MA in water .73

Fig 4.7 Experimental MSZWs of 70% mole percent of (S)-MA in water 74

Fig 4.8 Experimental MSZWs of (RS)-MA in water at different cooling rates .75

Fig 4.9 Ternary phase diagram of (S)-Kp and 0.9:1.0 (vol) mixture solvent of ethanol and water, ■, 15oC; ♦, 20oC; ▲, 25oC; ●, 30oC 79

Fig 4.10 Solubility and Metastable zone width of 50% and 75% mole 80

Fig 4.11 Effect chart of L9 design for (RS)-Kp .88

Fig 4.12 Effect chart of L9 design for 0.94 mole fraction of (S)-Kp and (S)-Kp 88

Fig 5.1 Concentration profiles of crystal suspension density MT, solute concentration C and supersaturation ∆C in kinetics measurement of (S)-MA 103

Fig 5.2 Concentration profiles of crystal suspension density MT, solute concentration C and supersaturation ∆C in kinetics measurement of (RS)-MA .103

Fig 5.3 Typical crystal size distribution in kinetic measurement of (S)-MA 105

Fig 5.4 Typical crystal size distribution in kinetic measurement of (RS)-MA .105

Fig 5.5 Typical s plane analysis to estimate crystal nucleation and growth rates for (S)-MA s f L2=0.1, G=3.25×10-8m/min, B= 4.98×108#./min.m3 .108

XII

Fig 5.6 Typical s plane analysis to estimate crystal nucleation and growth

Fig 5.7 Typical s plane analysis to estimate crystal nucleation and growth

Fig 5.8 Typical s plane analysis to estimate crystal nucleation and growth

Fig 5.9 Solubility of (RS)-Kp and (S)-Kp with different temperature .112 Fig 5.10 Crystal size distribution of (RS)-Kp in kinetic measurement of

(RS)-Kp .113 Fig 5.11 Crystal size distribution of (S)-Kp in kinetic measurement of (S)-

Kp 113

Fig 5.12 Typical s-plane analysis to estimate crystal nucleation and growth

Fig 5.13 Typical s-plane analysis to estimate crystal nucleation and growth

Fig 5.14 Typical s-plane analysis to estimate crystal nucleation and growth

Fig 5.15 Typical s-plane analysis to estimate crystal nucleation and growth

Fig 6.1 Partial separation of MA on Chiralcel AD-H semi-preparative

HPLC column (dimension 250mm L x 10mm I.D) at different

loadings 8.07mg and 16.15mg per injection using

Heptane/TFA/IPA (95/0.1/5 v/v) as mobile phase, at 25°C column

temperature, flow rate of 4.5ml/min and UV-Vis detection at

210nm .125Fig 6.2 Fraction collection under semi-preparative HPLC separation of

MA on Chiralcel AD-H column (dimension 250mm L x 10.00 mm

XIII

I.D.) under separation conditions: Heptane/TFA/IPA (95/0.1/5 v/v)

at 25°C column temperature, flow rate of 4.5ml/min and UV-Vis

detection at 210nm Fraction (a) collected at retention time 27-37

minutes and fraction (b) is collected at 37-43 minutes .126Fig 6.3 Chromatogram of two fractions (a) and (b) obtained through semi-

preparative HPLC separation of mandelic acid on Chiralcel AD-H

analytical column (dimension 250mm L x 4.6 mm I.D.) under

separation conditions: Heptane/TFA/IPA (95/0.1/5 v/v) at 25°C

column temperature, flow rate of 1.0 ml/min and UV-Vis detection

at 210nm 127Fig 6.4 Progression of direct crystallization of mandelic acid 129Fig 6.5 Three different cooling profiles .134Fig 6.6 The concentration of both enantiomers in the liquid phase and

corresponding process trajectory for controlled cooling process .135Fig 6.7 The concentration of both enantiomers in the liquid phase and

corresponding process trajectory for forced cooling process .136Fig 6.8 The concentration of both enantiomers in the liquid phase and

corresponding process trajectory for linear cooling process 137Fig 6.9 Calibration curve of optical rotation with concentration for pure

(S)-MA .138

Fig 6.10 Calibration curve of melting temperature with the mole percent

(S)-MA .139 Fig 6.11 DSC results for the final products and pure (S)-MA .140 Fig 6.12 HPLC results of final products and pure (S)- and (RS)-MA 140 Fig 6.13 Crystal size distribution of (S)-MA seeds and crystal products

from different cooling profiles 142Fig 6.14 Effect of seed size on the final CSD of preferential crystallization

for MA .144

Fig 7.1 Partial separation of Kp on Chiralcel AD-H semi-preparative

HPLC column (dimension 250mm L x 10mm I.D) at loadings

5.0mg per injection using hexane/IPA (90/10 v/v) as mobile phase,

at 25°C column temperature, flow rate of 3.5ml/min and UV-Vis

detection at 254nm 151Fig 7.2 Fraction collection under semi-preparative HPLC separation of Kp

on Chiralcel AD-H column (dimension 250mm L x 10.00 mm I.D.)

XIV

under separation conditions: hexane/IPA (90/10 v/v) as mobile

phase, at 25°C column temperature, flow rate of 3.5ml/min and

UV-Vis detection at 254nm.Fraction (a) collected at retention time

15.3-17 minutes and fraction (b) is collected at 17-22 minutes 152Fig 7.3 Chromatogram of fractions (b) obtained through semi-preparative

HPLC separation of ketoprofen on Chiralcel AD-H analytical

column (dimension 250mm L x 4.6 mm I.D.) under separation

conditions: hexane/IPA (90/10 v/v) as mobile phase, at 25°C

column temperature, flow rate of 0.8ml/min and UV-Vis detection

at 254nm 153Fig 7.4 The HPLC analyzing results for the crystal products of different

initial composition, (a) 92% mole percent (S)-Kp; (b) 94% mole

percent (S)-Kp; (c) 96% mole percent (S)-Kp .154

Fig 7.5 Cooling process to obtain pure enantiomer .156Fig 7.6 HPLC results of the final crystal products with different cooling

degree for ketoprofen: (a) Exp 01; (b) Exp 02; (c) Exp 03; (d) Exp

04; (e) Exp 05 158

XV

Nomenclature

Symbols

k G Growth rate constant

k v Crystal volumetric shape factor

origin

Greek Letters

1

Chapter 1 Introduction

2

Biological molecules often have the asymmetric carbon atoms, where each of the four bonds around the carbon atom is linked to a different atom (Rosanoff, 1906) Such molecules are called chiral molecules or racemic mixtures They exist in two enantiomorphic forms which are the mirror image of each other, but their mirror images are not superimposable with each other A pair of chiral enantiomorphic forms

is called enantiomers (Fig 1.1) The word enantiomer is derived from the Greek εναντιοζ (enantios), which means opposite A Molecule that is superimposable on its mirror image is called achiral The asymmetric carbon is called the chiral center or stereogenic center

Fig 1.1 A pair of chiral enantiomorphic forms

The two enantiomers of a racemic species differ only in the spatial arrangement The absolute configurations of chiral molecules are designated according to the Cahn-Ingold-Prelog convention It defines and applies a set of rules to assign the

designators R and S (from the Latin rectus, right, and sinister, left) to chiral carbon

3

atoms according to the sequence rule (Cahn et al., 1966) The oldest known manifestation of molecular chirality is the optical activity, rotatory power, the property that is exhibited by the rotation of the plane of polarization of light The two enantiomers of a given compound have rotatory powers of equal absolute value but of opposite sign, or sense One is positive, or dextrorotatory, while the other is negative,

or levorotatory The optical activities of enantiomers are designated with the optical

descriptor (+) or d (dextrorotatory), for clockwise rotation of polarized light, and (-)

or l (levorotatory) corresponding to anticlockwise rotation Another nomenclature uses Fisher projection formulas with D and L notions, mostly for amino acids and

sugars These terms do not provide any structural information about the molecules The configurational nomenclatures are discussed in more detail by Juaristi (1991) and Eliel et al (1994)

The two enantiomers have identical physical and chemical properties in an achiral environment because they exist in an identical chemical configuration, but they are subtly different in three dimensions conformation which gives rise to different biological properties (Walle et al, 1988, Williams and Lee, 1985) Enantiomers only behave differently in a chiral environment, such as biological system or a chiral medium (Ariëns et al., 1883; Eliel et al., 1994) This phenomenon is very important in the pharmaceutical industry

It is well known that very little is symmetrical in nature Indeed, most of nature

is chiral, including the human body (Wainer, 2001) The basic element of human body, such as proteins, nucleic acids and polysaccharides, lipids and steroids, possess chiral characteristic structures which are closely related to their functions It is interesting to

notice the homo-chirality of human body where proteins consist exclusively of amino acids and nucleic acids of D-sugars, respectively (Jacques, 1993) According to

L-4

the accepted theory, enzymes and other biological molecules inside human body are chiral molecules that bind selectively to certain molecular structures and thus show the enantioselective in their binding to the messenger molecules The lock and key model for enzyme and receptor interactions also predicts that only an exact

stereochemical structure fits to the corresponding site In fact, the D-amino acids are recognized by enzymes, receptors and other biological molecules, whereas the L-

forms would be rejected in the same reaction

As the human body is a highly stereospecific environment, it has long been known that different enantiomers may show very different biological activities in their pharmacological and toxicological effects, since they interact with biological macromolecules, the majority of which are stereoselective (Drayer, 1986; Wainer, 1993) Strong emphasis has been concentrated upon the search of therapeutic benefits with the goal of developing safer and more effective drugs The high degree of stereoselectivity of many biological processes implies that when a given racemic mixture is administered as a drug both enantiomers should not have to be equally potent In fact, very often one of them represents the more active isomer for a given action (eutomer), while the other one (distomer) might be even active in a different way, contributing to side-effects, displaying toxicity, or acting as antagonist (Wainer,

1993 and Maier et al., 2001) One Classical example is “the thalidomide disaster” of

later 1950s The drug was marketed as the racemate; it was found later that the enantiomer showed the desired hypnotic potency whereas the S-enantiomer caused

R-serious teratogenic effects (Ockenfels et al., 1976) In the review, Wainer (2001) used some examples to show how using active enantiomers as therapeutic agents may yield several benefits, including more predictable pharmacokinetics, more accurate drug monitoring and enhanced tolerability As a result of these benefit, the therapeutic use

separation these drugs since the target receptors do not distinguish between the two chemical entities There are few drugs in this category

properties: most racemates drugs belong to this category Only one of the enantiomers has the desired effect, where the other enantiomer may inactive or cause undesired side effects

provide better alternatives to their respective racemates

enantiomers of the racemate drug can be separated to give two different types of drugs together Each drug has different therapeutic effect

As mentioned above, it is very important to well understand the pharmacological, pharmacokinetic, therapeutic as well as toxicological effect of both enantiomers in order to know whether the pure enantiomer has better, same or worse therapeutic effect compared with its racemate It is necessary to develop effective therapeutic agents in optically pure form in order to fully exploit the nature and to evaluate the enantiomeric purity and toxicity/activity of any existing or new drugs However because of the limitation of the methods for production of single

The FDA’s requirements have stimulated the development of novel techniques for asymmetric synthesis and enantiomeric separation in pharmaceutical industry Especially, for the case 2 and 4 group chiral drugs, obtaining the pure enantiomer as commercial drugs is necessary for the better therapeutic effect So in recent years, enantioselective production of chiral drugs has continued to grow at a rapid pace (Waldeck, 1993; Stinson, 2001; Rouhi, 2002)

With increasing awareness of need for higher drug specificity, the worldwide market for chiral drugs grew 11% over the previous year to $96 billion in 1998 Also, Single-enantiomer drug sales show a continuous growth worldwide (21% sales increment from 1997 over 1996) and many of the top-selling drugs are marketed as single enantiomer (269 of the top 500 drugs) (Stinson, 1997, 1998, 1999) In addition, between 1992 and 2002, the world market for optically pure chemical compounds increased from $30 billion to an estimated $100 billion According to a survey, the global sales of single enantiomer compounds are expected to reach about $15 billion

by the end of 2009, growing annually by 11.4 % (Rouhi, 2004)

7

With the development of the markets for pure enantiomer chiral drugs, many people are looking for some new enantioselective technologies to obtain the pure enantiomer by various approaches Currently, there are several methods for obtaining pure enantiomer, which can be broadly classified into two categories (Sheldon, 1993): the first, the direct synthesis of optically pure compounds; the second method involves obtaining pure enantiomer from separation of racemate species which have been easily synthesized As racemate synthesis is easier, cheaper and less time consuming than the single isomer synthesis, the separation method is popular in industry

According to the regulatory requirements and the consideration for a better therapeutic effect, these chiral separation technologies will continue to be a very promising area which is full of challenges Therefore this thesis focuses on the enantiomer separation to obtain the desired pure enantiomer of chiral drug There are several widely used chiral separation methods which can be selected, such as chiral enzymatic separation, chiral chromatography and chiral crystallization Each of them has its own advantages and inadvantages The detail background and application about each chiral separation method will be introduced in the literature review of the next chapter Based on analyzing advantages and inadvantages of these methods, the promising method of coupling the two different separation methods, such as direct crystallization and chromatography, would be proposed and the direct crystallization process for racemic compound was mainly studied in order to obtain pure product with the good quality in this dissertation

8

Chapter 2 Literature review

9

The industrial production of pure enantiomers may involve either resolution or stereoselective synthesis The stereoselective (asymmetric) synthesis uses the chemical reaction of an enantiomeric agent or catalyst with a substrate to produce a single isomer This process may be uneconomical because of the low yield or high cost of the enantiomeric reagents Consequently, resolution of racemates (chiral separation) is still the leading technology in the commercial production of enantiomerically pure chiral substances (Li, 1997)

Generally, the often used chiral separation method can be classified into three types: enzymatic separation, separation by chiral chromatography, and chiral crystallization Table 2.1 lists some important resolution methods

Table 2.1 Resolution methods

Enzymatic separation

This process uses stereoselective biological activity to resolve single enantiomers

Separation by chiral

chromatography

This procedure utilizes columns with a chiral stationary phase from which the isomers are separated through

diastereomeric interactions

Direct crystallization

One enantiomer is selectively crystallized from a racemic solution controlled by either thermodynamic or kinetic factors

Crystallization

Diastereomeric crystallization

This process involves a reaction of the racemate with an optically pure acid or base (the resolving agent) to give a mixture of two diastereomer salts whose physical properties are different