NOVEL CHIRAL STATIONARY PHASE FOR THE ENANTIOSEPARATION OF RACEMIC DRUGS LO MEE YOON (MSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements I would like to express my immense gratitude to my supervisor, A/P Ng Siu Choon for his guidance and supervision throughout the project. I would also like to thank my co-supervisor, Prof Ching Chi Bun for his support. I would like to thank my parents and sisters for their advice and encouragement during my studies. I would also like to thank my boyfriend, kinseng for his understanding and continual support. I would like to thank all my friends in the functional polymer groups, Dr Chen Lei, Dr I Wayan, Yeang Chyn, Yi Fei, Teck Chia, Yin Fun, Xiang Hua, Zou Yong, Wei Hua and Daming for their kind support. Especially thanks to Frances for her kind assistance. Last but not the least; I would like to thank National University of Singapore for the award of the Research Scholarship. i Table of Contents Acknowledgements………………………………………………………….i Table of Contents………………………………………………………….ii Summary…………………………………………………………………vii Abbreviations and Symbols……………………………………………….x Chapter Introduction 1.1 Overview 1.2 Importance of Chirality 1.3 Historical Review 11 1.4 High Performance Liquid Chromatographic (HPLC) 14 1.4.1 Separation Modes in HPLC 15 Chromatographic Methods 16 1.5.1 Indirect Chromatographic Method 16 1.5.2 Direct Chromatographic Method 17 1.6 Cyclodextrin and Chiral Stationary Phase 26 1.7 Three-Point Interaction − Origin of CSP 33 1.8 The Supporting Matrix for CSPs 34 1.9 Detector 38 1.5 ii 1.10 Scope of Work 39 References 41 Chapter Synthesis of Ether-linkage β-CD Bonded CSPs 49 2.1 Introduction 50 2.2 Selective Modification of β-CD 51 2.2.1 Preparation of 5-Iodo-hex-1-ene and 11-Iodo-undec-1-ene 52 2.2.2 Synthesis of 2a, 2b and 2c 53 2.3 Synthesis of Perfunctionalized Carbamolylated β-CD 3a-3f 55 2.4 Hydrosilylation and Immobilization 55 2.5 Synthesis of CSP 5g 57 2.5.1 Preparation of Hydride-modified Silica Gel 58 2.5.2 Immobilization of 2a onto 60 Summary 61 2.6 References 62 Chapter Enantioseparations under Normal and Reversed Phase 64 3.1 Introduction 65 3.2 Simple Racemic Compounds 67 3.3 Flavanone Compounds 70 iii 3.3.1 Normal Phase 72 3.3.2 Reversed Phase 76 3.4 β-Blockers 79 3.5 Acidic Compounds 82 3.6 Conclusion 83 References 84 Chapter Effects of Substituents under Reversed Phase 86 4.1 Introduction 87 4.2 Enantioseparations under Reversed Phase 90 A. Carbonyls 90 B. Alcohols 92 C. Amines 94 D. β-Blockers 96 E. Flavanones 98 F. Weak Acids 99 4.3 G. Racemic Drugs 100 Conclusion 103 References 105 Chapter Effects of Spacer Length 106 iv 5.1 Introduction 107 5.2 Normal Phase 109 5.3 Reversed Phase 114 5.4 Conclusion 116 References 118 Chapter Optimization of Enantioseparation Conditions and Chromatographic Properties 119 6.1 Introduction 120 6.2 Mobile Phase Composition 121 6.2.1 Normal Phase 121 6.2.2 Reversed Phase 122 6.3 Effects of pH under Reversed Phase 124 6.4 Flow Rate 126 6.5 Organic Modifier under Reversed Phase 128 6.6 Concentration of TEAA Buffer under Reversed Phase 129 6.7 Loading Capacity 130 6.8 Thermodynamics Study 133 6.8.1 Theory 134 Conclusion 137 6.9 v References 138 Chapter Experimental 139 7.1 General 140 7.1.1 Materials 140 7.1.2 Instrumentation 140 7.1.3 Packing of CSPs 141 7.1.4 HPLC Instrumentation 141 7.1.5 Basic Chromatography Parameters 142 7.2 Preparation of Ether-linkage CSPs 143 7.3 Synthesis of Perfunctionalized Carbamolylated β-CD 147 7.4 Hydrosilylation and Immobilization 150 7.5 Synthesis of CSP 5g 151 7.6 Packing of CSPs 152 Chapter Conclusions and Suggestions for Future Work 153 8.1 Conclusion 154 8.2 Suggested Future Works 156 List of Publications 157 vi Summary The importance of chirality is attracting more attention from researchers nowadays. Purification of enantiomers is always preferred for safer clinical applications particularly in biomedical and pharmaceutical areas. Chiral drugs are generally administered either as enantiomers or as racemates, and very often two enantiomers of the same racemate possess different pharmacological effects. In order to avoid side effects that could be caused by the presence of an undesirable component in a racemic drug, it is important to design a method for effective separation of racemates. The separation of enantiomers is widely studied in analytical chemistry, especially in the area of high performance liquid chromatography (HPLC) due to its versatility. In this work, series of ether-linkage β-cyclodextrin (β-CD) bonded chiral stationary phases (CSPs) were synthesized. The CSPs were prepared by hydrosilylation and immobilization via a stable ether-linkage onto the surface of silica gel. The welldefined CSPs were characterized and packed into HPLC analytical (with –L) or microbore (without –L) columns for the analysis of racemic compounds or drugs. The synthesized CSPs were evaluated under normal and reversed phase HPLC. Selected chiral compounds and drugs were chromatographed on the CSPs for the investigation of possible separation mechanisms. Nonpolar solvents such as hexane and isopropanol (IPA) were used as mobile phase under normal phase. H-bonding and π-π interactions dominate under normal phase. Nonpolar solvents tend to occupy the CD vii cavity and preventing inclusion complexation from taking place. Under reversed phase, polar solvents such as water, triethylammonium acetate (TEAA) buffer, methanol (MeOH) and acetonitrile (ACN or CH3CN) were used. Under reversed phase, inclusion complexation takes place. In this work, six β-CD bonded CSPs (ETHE-3PC, ETHE-6PC, ETHE-11PC, ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC) were conveniently prepared and characterized. Enantioseparation of selected racemic compounds was evaluated on phenylcarbamate CSP ETHE-3PC-L which was packed into a HPLC analytical column [∅ 4.6 x 250 mm] under normal and reversed phase. The CSP was proven to be a generic CSP to resolve a wide range of structurally diverse racemic compounds. Three CSPs with the same spacer length, ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC were prepared. The CSPs were modified with different perfunctionalized substituents on the remaining C2-, C3- and C6- CD hydroxyl groups. Chromatographic results for the three CSPs were compared with ETHE-3PC for the investigation of the effects of substituents on enantioseparations. All four CSPs were packed into HPLC microbore column [∅ 2.1 x 150 mm]. Naphthylcarbamate CSP ETHE-3NC showed good discriminating abilities towards amines and β-blockers; 4-methoxyphenylcarbamte CSP ETHE-3pMPC showed good enantioselectivity particularly for β-blockers. ETHE3pCPC on the other hand, showed high enantioselectivity for weak acids, carbonyls, alcohols, flavanones and other electron-rich analytes. However, the three CSPs failed to perform satisfactory enantioseparation under normal phase. viii Phenylacarbamte CSP ETHE-3PC, ETHE-6PC and ETHE-11PC were prepared in different spacer lengths for the study of the effect of spacer length on the enantioseparation abilities of the CSPs. Different spacer lengths afforded different chiral discrimination behaviors, particularly on solute retentivity and selectivity. There appears an optimum spacer length which seems to be close to methylene groups under normal and reversed phase. Lastly, study on the optimization of chromatographic conditions such as mobile phase composition, flow rate, buffer concentration and pH, and selection of polar organic modifiers were included. Thermodynamics and surface loading studies were also investigated. ix The electron-withdrawing group (−Cl) present on the phenyl ring can interact with πsuccessive compounds. Due to the lack of favorable H-bonding groups, ETHE-3pCPC also failed to afford satisfactory enantiorecognition abilities under normal phase. In this chapter, for better understanding of the effects of substituents, the chiral discrimination behaviors of ETHE-3NC, ETHE-3pCPC and ETHE-3pMPC were compared with ETHE-3PC on selected samples under reversed phase. 4.2 Enantioseparations under Reversed Phase A. Carbonyls From Table 4.2, we observed the highest k’ values and enantioselectivity on ETHE-3pCPC. Carbonyls are electron-rich compounds (Figure 4.2) and in addition to electron-donating groups such as –OMe, –NH2 and an electron-inductive alkyl chain attached on the analytes, the carbonyl compounds are capable of undergoing stronger interaction (π-donor/π-acceptor interaction) with ETHE-3pCPC as a result of the CSP’s π-deficient characteristics. O N C H O - + N C H Figure 4.2. Resonance of an amide bond. Since strongest solute retention was observed for ETHE-3pCPC, analysis on ETHE-3pCPC was performed at a lower pH value for faster elution of the solutes. 90 Lower pH helps to ionize the analytes and gives shorter retention time due to reduced solute hydrophobicity. All the carbonyls were resolved on ETHE-3pPC except for prilocaine. It is likely that the two –NH– groups in prilocaine undergo intramolecular Hbonding and hinder the chiral interactions between ETHE-3pCPC and the solute. Analytes with para-substituted electron-donating groups such as –NH2 and –OMe (e.g. aminoglutethimide and formoterol) helps to improve enantioselectivity on ETHE3pCPC. Table 4.2. Separation of compounds containing unsaturated carbonyl groups. Compound k’1 k’2 α Rs Thalidomide 0.88 0.39 2.00 0.43 1.46 1.95 3.48 0.74 1.67 1.15 1.74 1.70 2.00 1.25 2.87 1.17 ETHE-3PC 0.88 1.04 3.25 2.08 2.04 2.57 2.00 3.11 1.27 ETHE-3PC O O H N O N O Prilocaine Me O N H 0.56 HN Aminoglutethimide O H2N Et N H O 5.30 Formoterol H N OH MeO NH O 8.60 4.69 - ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3NC ETHE-3pCPC 3.62 1.21 2.12 1.17 - OH H 6.48 - CSP ETHE-3pMPC ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3PC 1.13 0.75 ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: 1% TEAA pH 5.45/MeOH=60/40 (v/v); at pH 4.10; flow rate at 0.2 ml/min and UV detection at 254nm. 91 Thalidomide was resolved on all the six CSPs. The unsaturated carbonyl and amine groups provide additional sites for H-bonding. π-π Interaction was favored with the presence of a phenyl ring attached. Furthermore, the analyte is bulky for the formation of a tighter and more stable inclusion complex. Figure 4.3. Effects of substituent on k’, α and Rs for the enantioseparation of thalidomide (a = separation factor). B. Alcohols In Table 4.3, catechin and normethanephrine are electron-rich analytes with the presence of more than one substituted electron-donating methoxyl or hydroxyl groups. These electron-rich solutes were well resolved on ETHE-3pCPC due to stronger πdonor/π-acceptor and dipole interactions between the solutes and the π-deficient CSP. The para-hydroxylated octopamine was also resolved on ETHE-3PC though stronger 92 retention and higher enantioselectivity were afforded by ETHE-3pCPC as expected. None of the three analytes were resolved on ETHE-3NC and ETHE-3pMPC mainly due to steric repulsive interactions between the electron-rich solutes and π-successive CSPs. Table 4.3. Separation of alcohols under reversed phase. Compound k’1 Catechin α Rs - OH OH O HO k’2 ETHE-3PC - 0.78 0.91 1.17 0.50 - OH CSP ETHE-3NC ETHE-3pCPC ETHE-3pMPC OH Normethanephrine OH NH2 MeO 7.52 HO Octopamine 0.41 OH NH2 HO 0.48 16.13 0.67 0.78 - ETHE-3PC 2.14 2.00 1.60 1.14 1.64 1.00 ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: 1% TEAA pH 5.45/MeOH=60/40 (v/v); at pH 4.10. For adrenergic β-receptor agonists in Table 4.4, most of the analytes were separated on ETHE-3pCPC and ETHE-3PC. Steric hindrance may be the main factor for the loss of enantioseparation of such compounds on ETHE-3NC and ETHE-3pMPC as mentioned earlier. ETHE-3pCPC was able to resolve all three compounds. It appears that ETHE-3pCPC is capable of resolving highly electron-rich solutes. 93 Table 4.4. Separation of β-receptor agonists on ETHE-3PC and/or ETHE-3pCPC. Compound k’1 k’2 α Rs Albuterol 0.17 0.25 1.50 0.43 ETHE-3PC 1.67 0.50 ETHE-3NC ETHE-3pCPC ETHE-3pMPC 2.35 3.76 ETHE-3PC 2.13 ETHE-3NC ETHE-3pCPC ETHE-3pMPC OH H N - 0.13 HO - CH2OH Clenbuterol OH Cl 1.29 H N 1.00 H2N Cl Terbutaline OH HO H N 0.18 OH 0.22 3.04 2.21 0.45 - 2.21 CSP ETHE-3PC 3.99 1.30 ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: at 1% TEAA pH 5.45/MeOH=60/40 (v/v); at pH 4.10. C. Amines Almost all amine compounds tabulated in Table 4.5 were separated on ETHE- 3NC and ETHE-3PC (with the exception of quinocide). The five amine-containing compounds are hydrophobic and bulky with the presence of more than one ring. This explains the strong solute retentivity and good enantioselectivity of the five compounds on ETHE-3NC. As stated earlier, bulkier solutes are capable of forming tighter inclusion complexes with ETHE-3NC due to its wider CD rim and higher hydrophobicity. It appears that amines interact more effectively with the phenyl or naphthalene ring in the CSPs. 94 ETHE-3pMPC was able to resolve ionic compounds such as laudanosine methiodide and triazine. The positively charged amine could undergo dipole interaction and π-donor/π-acceptor interaction with the methoxyl group of the π-conjugated ETHE3pMPC. Laudanosine methiodide and triazine were not resolved on ETHE-3pCPC as expected. Table 4.5. Separation of amine-containing compounds under reversed phase. Compound Laudanosine methiodide OMe k’1 k’2 α Rs 7.50 10.48 9.21 13.26 8.69 1.53 1.27 1.23 1.27 1.38 1.46 3.33 2.83 1.91 2.84 1.09 1.74 1.29 1.52 1.70 OMe MeO + N 5.95 CSP ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC MeO Triazine Cl NH2 N H2N 1.92 2.95 N + NH 1.17 Ph Pheniramine N CH3 NH2 HN ETHE-3NC ETHE-3pCPC ETHE-3pMPC 2.67 5.08 3.08 5.83 - 1.00 0.80 1.16 1.14 ETHE-3PC 4.75 9.26 6.58 12.04 - 1.00 1.05 1.38 1.30 ETHE-3PC 0.72 1.18 N Primaquine ETHE-3PC N ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3NC ETHE-3pCPC ETHE-3pMPC MeO Quinocide NH2 HN N MeO CH3 8.31 9.31 - ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: at 1% TEAA pH 5.45/MeOH=60/40; at pH 4.10. 95 Though structurally similar, primaquine and quinocide exhibit different enantioseparation results under the same conditions. From Table 4.5, we observed that the chiral center of quinocide is further away from the aromatic ring. Unlike primaquine, the chiral center is placed one atom away from the aromatic ring. Resonance of πelectrons and the presence of a sp2-hybridized carbon closer to the chiral center in primaquine help to enhance its chiral recognition on the CSPs. D. β-Blockers ETHE-3pMPC was capable of resolving all seven test β-blockers in Table 4.6; five were resolved on ETHE-3NC and four on ETHE-3PC. ETHE-3pCPC on the other hand, was able to resolve only alprenolol. Though fewer β-blockers were resolved on ETHE-3PC, the CSP generally affords better selectivity than ETHE-3NC and ETHE3pMPC. The strongest solute retentivity was generally afforded by ETHE-3PC except for propanolol. Propanolol can undergo more favorable inclusion complexation with ETHE-3NC due to the presence of a hydrophobic and bulky naphthalene ring. β-Blockers were better recognized by ETHE-3NC and particularly ETHE3pMPC due to a wider CD cavity of the two CSPs. We observed that all the β-blockers in Table 4.6 are bulky solutes substituted with a flexible or a long chain. The larger CD rim in the two CSPs can trap the solutes more effectively to afford higher discriminating energy. 96 Table 4.6. Separation of β-blockers under reversed phase. Compound Acebutolol O OH k’1 k’2 α Rs 1.67 1.45 1.67 1.29 2.42 1.67 1.29 0.86 ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC 3.46 1.87 2.62 1.78 6.45 2.95 3.50 2.78 1.87 1.58 1.33 1.56 4.00 0.44 0.54 1.61 ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC 1.35 1.34 1.30 0.67 ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC 1.57 1.49 ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC 1.55 1.31 1.30 0.60 1.33 0.71 1.38 1.00 1.42 1.00 1.91 1.15 1.62 0.50 1.56 0.67 H N O O N H Alprenolol OH H N O 0.96 1.17 1.21 4.91 7.69 Atenolol OH O O 0.82 H N H2N Betaxolol OH O O Metoprolol OH H N O O H N 3.58 2.30 2.00 Pindolol OH O 1.73 H N N H 1.74 Propanolol OH O H N 6.00 11.08 4.17 5.54 3.00 2.67 2.39 2.48 11.29 12.75 6.50 CSP ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: 1% TEAA pH 5.45/MeOH=60/40 (v/v). at pH 4.10; at 80/20 (v/v), pH 4.10. 97 E. Flavanones Though having a naphthalene ring as potential π-π interaction and inclusion complexation sites, ETHE-3NC failed to perform chiral discrimination on flavanones under specified conditions. The enantioseparation results were also not satisfactory in the presence of organic modifiers such as MeOH and CH3CN. However, the flavanones were easily resovled on ETHE-3PC, ETHE-3pCPC and ETHE-3pMPC. ETHE-3PC affords the highest enantioselectivity for most of the flavanones. Generally, retention for methoxylated flavanones was observed to be stronger than the hydroxylated derivatives as discussed in Chapter 3. From Table 4.7, we observe that for all the flavanones separated, the largest k’ values were afforded by ETHE-3pCPC. Strong retention is mainly due to π-donor/πacceptor interacting forces between solutes’ electron-donating groups (–OMe and –OH) and the π-deficient ETHE-3pCPC. This also explains the lower k’ values observed for the parent flavanone compared to substituted flavanones on ETHE-3pCPC due to the lack of an electron-donating group. Table 4.7. Separation of flavanones under reversed phase. Compound k’1 k’2 α Rs Flavanone 2.75 4.42 10.12 1.92 1.61 3.43 1.11 1.28 0.90 1.12 O O 9.12 1.50 CSP ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC 98 Table 4.7. (Continued) Compound k’1 k’2 α Rs 4’-hydroxyflavanone 1.58 2.42 6.88 1.12 1.63 2.80 ETHE-3PC 1.60 1.29 0.70 0.73 ETHE-3NC ETHE-3pCPC ETHE-3pMPC 2.12 5.08 1.58 1.31 1.93 ETHE-3PC 1.18 1.41 1.33 1.21 ETHE-3NC ETHE-3pCPC ETHE-3pMPC 5.17 2.75 1.40 2.44 ETHE-3PC OH O 4.29 0.88 O 6-hydroxyflavanone 1.63 O 4.29 1.12 HO O 4’-methoxyflavanone 3.71 OMe O 2.21 O 6-methoxyflavanone 5.08 O MeO O 7-methoxyflavanone MeO 18.00 3.08 4.58 O O 11.92 3.54 7.25 19.80 4.12 7.62 13.04 3.42 CSP ETHE-3NC ETHE-3pCPC ETHE3pMPC ETHE-3PC 1.25 1.04 1.43 2.94 1.10 1.34 0.76 1.46 1.66 4.00 ETHE3pMPC ETHE-3PC 1.09 1.34 0.67 1.23 ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3NC ETHE-3pCPC Condition: MeOH/water = 60/40 (v/v); flow rate at 0.2ml/min. F. Weak Acids Some chiral analytes can behave as a Lewis acid that provides electron pairs during the process of structural transformation in certain solvent system although they not have an acidic group in the molecule. These analytes such as althiazide and p-anisoin can possibly undergo epimerization in acidic medium. 99 Table 4.8. Enantioseparation of weak acids. Compound k’1 Althiazide H N Cl S O H2N S O 8.50 NH S O O p-Anisoin OH MeO OMe O 1.74 3.69 2.25 k’2 11.08 - 4.00 5.43 3.17 α 1.30 2.30 1.47 1.41 Rs 0.77 1.33 3.06 1.92 CSP ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: 1% TEAA pH 5.10/MeOH=65/35 (v/v); at pH 4.10. In Table 4.8, both weak acids were strongly retained and resolved on ETHE3pCPC. Althiazide has a chiral carbon next to a –NH– group and a – SO2NH– group, the delocalization of the electrons favors π-donor/π-acceptor interaction with the CSP ETHE-3pCPC. p-Anisoin was not resolved on ETHE-3PC but achieved satisfactory enantioseparation on the other three CSPs. It appears that ETHE-3PC is not good at separating weak acids, though separation of acidic compounds such as ofloxacin and 2phenylbutyric acid was previously reported in Chapter 3. G. Racemic Drugs Atropine belongs to the tropane alkaloid family that can be obtained from various solanaceous plants. The chiral recognition of atropine is mainly affected by the solute rigidity. From Table 4.9, it appears that chiral discrimination of the bridging structure of 100 atropine is not favored on ETHE-3NC. Atropine was resolved on the other three CSPs and highest selectivity was afforded by ETHE-3PC. Table 4.9. Enantioselectivity of atropine under reversed phase. Compound k’1 k’2 α Rs Atropine 1.75 3.78 2.91 1.78 5.67 2.12 1.24 1.56 1.24 1.56 O H3C N O OH 2.30 1.17 CSP ETHE-3PC ETHE-3NC ETHE-3pCPC ETHE-3pMPC Condition: 1% TEAA pH 5.10/MeOH=65/35 (v/v); at pH 4.10. Table 4.10 summarizes chromatographic results for chiral drugs that can be separated only on ETHE-3pCPC under specified conditions. Almost all the separated drugs possess electron-donating characteristics with the presence of π-conjugated substituents or atoms to undergo favorable π-donor/π-acceptor interaction with ETHE3pCPC. Table 4.10. Chiral drugs separated on ETHE-3pCPC. Compound Chromatographic results Omeprazole O N NH MeO Me N S OMe Me k’1 = 2.62 k’2 = 3.21 α = 1.22 Rs = 0.75 k’1 = 8.21 k’2 = 11.43 α = 1.39 Rs = 1.45 k’1 = 5.48 k’2 = 11.30 α = 2.08 Rs = 1.42 Trans-stibene oxide O Ph Ph Tetramisole N S N Ph 101 Guaiacol glyceryl ether k’1 = 11.56 k’2 = 11.96 OH O OH α = 2.19 Rs = 105 OMe Condition: 1% TEAA pH 4.10/MeOH=65/35 (v/v). Dropropizine, lansoprazole and diperodon were resolved on ETHE-3PC and ETHE-3pMPC (Table 4.11). It appears that the two CSPs are capable of resolving piperazines and imidazoles. The carbamate groups on the diperodon can H-bond to the CSPs’ carbamate linkage and stronger solute retention was generally observed for ETHE-3PC. The three chiral drugs were not resolved on ETHE-3NC and ETHE3pCPC. Table 4.11. Chiral drugs separated on ETHE-3PC and ETHE-3pMPC. Ph Compound k’1 k’2 α Rs Dropropizine 7.42 8.17 1.10 0.50 ETHE-3PC 6.50 8.17 1.26 0.78 ETHE-3pMPC 6.42 7.33 1.14 1.09 ETHE-3PC 3.00 4.39 1.46 1.43 ETHE-3pMPC 8.20 8.79 1.07 0.45 ETHE-3PC 5.62 6.33 1.12 0.75 ETHE-3pMPC OH N N CSP OH Lansoprazole N O N S CF3 O Me NH Diperodon O O N N H Ph O N H Ph O Condition: 1% TEAA pH 5.10/MeOH=65/35 (v/v). 102 4.3 Conclusion In this chapter, we have investigated the effects of phenylcarbamate, naphthylcarbamate, 4-chlorophenylcarbamate and 4-methoxyphenylcarbamte as chiral selectors in the enantioseparation of the selected chiral compounds or drugs under reversed phase. It appears that substituents on chiral selectors play an important role in influencing the enantioseparation of enantiomers. In conclusion, phenylcarbamate CSP ETHE-3PC is the most commonly used CSP for the enantioseparation of a wide range of structurally diverse racemic compounds. Its simplicity in structure makes it easier for the study of separation mechanisms. Though it is capable of resolving racemic compounds under normal and reversed phase, it failed to perform separation on structurally or functionally complicated analytes such as althiazide and quinocide. Some analytes require additional π-basic or π-acidic sites, or larger CD cavity, or higher hydrophobicity, or even greater steric interactions to be resolved. Naphthylcarbamate CSP ETHE-3NC is higher in hydrophobicity and rigidity due to the presence of a bulky naphthalene ring. The CSP is generally not good for discriminating alcohols and flavanones. However, it showed good discriminating abilities towards amines and β-blockers. Another π-conjugated CSP prepared in this work is the 4-methoxyphenylcarbamate CSP ETHE-3pMPC. ETHE-3pMPC showed good enantioselectivity on imidazoles, piperazines, flavanones and particularly β-blockers. It 103 affords better chiral recognition abilities on electron-deficient analytes due to its πconjugated characteristics. However, ETHE-3pMPC failed to perform satisfactory enantioseparation on test alcohols and amine compounds. 4-Chlorophenylcarbamate CSP ETHE-3pCPC on the other hand, showed high enantioselectivity towards highly electron-rich analytes as a result of its π-deficient characteristics. The CSP appears to be not good for chiral discrimination on β-blockers. Yet, it showed good enantioselectivity on electron-rich analytes such as weak acids, carbonyls, alcohols, flavanones, ethers, epoxides, thiazoles and imidazoles. We understand that there is no single CSP that can have universal enantioselectivity abilities to resolve all types of chiral compounds. In this work, the general enantiorecognition trend for each CSP was deduced. 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(BSA) for chiral resolution.70 19 74 Blaschke: Synthesis of chiral polymers. 71 1975 Cram et al: Chromatography with chiral crown ethers 19 77 Use of chiral chemical shifts reagent for chiral LC 19 79 Pirkle and House: Synthesis of first silica-bonded CSP.58 Okamoto et al: Synthesis of helical polymer for chiral LC.72 19 81 First commercially available chiral HPLC phase. 64 19 82 Allenmark: Use of agarose-bonded... important to determine the stereoisomeric composition of chemical compounds, especially of pharmaceutical significance .13 Unfortunately, there are many racemic drugs where the stereospecificity of the metabolism and/or the pharmacodynamic effects of the enantiomers are still unknown .14 -16 Today, about 56 % of the synthetic drugs1 4, 17 currently in use are chiral compounds, out of these chiral pharmaceuticals,... enantiomers and their sales accounted for 52 % (US$ 71. 1 billion) of the total US $13 5.9 billion.3, 18 -19 Since chiral compounds represent more than 50 % of the worldwide most frequently prescribed drugs, the interest in the preparation and isolation of chiral drugs has increased dramatically However, in spite of the knowledge that the specific effect of a drug is often caused by just one enantiomer, racemic. .. late 19 61, thalidomide was found to be a dangerous drug for pregnant women, where the d-form is a safe sedative but the l-form causes severe birth defects and deformities (Figure 1. 6) Arising from the increasing pressure exerted by the scientific community towards restricting the use of chiral drugs in their racemic form, the Food & Drug Administration of the U.S (FDA) began to regulate the marketing of. .. the most useful means for chiral separation.78-80 14 In HPLC, the characteristics of both the stationary phase and the mobile phase can affect the selectivity and performance The variety of these selective interactions can be increased by suitable chemical modification of the silica surface Therefore one can say that HPLC is a more versatile and powerful method than other chromatographic methods. 81- 84... a chiral molecule that is bound to the stationary phase The separation is due to differences in energy between temporary diastereomeric complexes formed between the solute isomers and the CSP; the larger the difference, the greater the separation The observed retention and efficiency of a CSP is the total of all the interactions between the solutes and the CSP, including achiral interactions 17 1. 5.2.a... it is inconvenient for preparative applications because the chiral additive must be removed from the enantiomeric solutes 1. 5.2.b Chiral Stationary Phases At present, there are over a hundred of CSPs for HPLC that are commercially available According to Wainer33, there are five major classes of HPLC CSPs based on the type of analyte-CSP complexes formed The Type 1 or “Pirkle” phase forms analyteCSP complexes... Racemic drugs can cause problems because of the differences not only in the biological effects but also in the pharmacokinetics of the enantiomers as stated by Caldwell in the 19 80s, The racemate killed a number of people who had accumulated gram quantities of the enantiomer that was more slowly metabolized” Table 1. 1 Examples of Pharmaceutical and Food and Drink products that show the effect of chirality... and chemical bonding onto the surface of silica gel through a spacer as depicted in Figure 1. 9 This chemical bonding makes the column more stable in the aqueous phases These CSPs are commercially available and are widely used for separation of a variety of drugs and 21 amino acids .10 0 -10 1 The separation is relatively good under normal phase, polar organic phase and reversed phase R R R R R R Silica... which slow down movement of the solute having higher affinity for the stationary phase Due to the cumulative nature of the chromatographic separation, it is trivial practice to completely resolve a mixture of two components that differ in the free energy of interaction with the stationary phase by as little as 0.025 kJ/mol, which corresponds to the column selectivity value of α = 1. 01 In addition, as a . NOVEL CHIRAL STATIONARY PHASE FOR THE ENANTIOSEPARATION OF RACEMIC DRUGS LO MEE YOON (MSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY. G. Racemic Drugs 10 0 4.3 Conclusion 10 3 References 10 5 Chapter 5 Effects of Spacer Length 10 6 iv 5 .1 Introduction 10 7 5.2 Normal Phase 10 9 5.3 Reversed Phase 11 4 5.4 Conclusion 11 6. Capacity 13 0 6.8 Thermodynamics Study 13 3 6.8 .1 Theory 13 4 6.9 Conclusion 13 7 v References 13 8 Chapter 7 Experimental 13 9 7 .1 General 14 0 7 .1. 1 Materials 14 0 7 .1. 2 Instrumentation 14 0