ENANTIOMERIC RESOLUTION OF SOME PHARMACEUTICALS BY LIQUID CHROMATOGRAPHY

Abstract

The importance of chirality of the molecules has been recognized and is required to be addressed for biologically active compounds, in the areas dealing with pharmaceuticals, agrochemicals, food analysis, fragrances, chiral pollutants, asymmetric synthesis, clinical and forensic evaluation. This interest can be attributed largely to the fact that the enantiomers may have different pharmacological activities, as well as different pharmacokinetic and pharmacodynamic effects in chiral systems. Therefore, development of efficient analytical methods for enantioseparation has become an essential part of the drug development process. Among the various available methods for obtaining enantiomerically pure compounds, liquid chromatographic (LC) techniques particularly high performance liquid chromatography (HPLC) is extensively used for resolution of a variety of chiral biochemicals. Present thesis deals with studies on direct and indirect enantioseparation of certain chiral carbonyl compounds, J3-amino alcohols, proteinogenic-a-amino acids, se lenomethionine, 3-blockers, (RS)-ketorolac and (RS)-amlodipine. These chiral compounds/ drugs have wide applications in the field of chemistry, biochemistry, medicine, etc. The enantiomcric separation has been carried out using different chiral derivatizing reagents (CDRs), chiral stationary phases (CSPs) and chiral selectors. First chapter deals with preamble to present studies including importance of chirality in pharmaceuticals, and brief introduction to the applicable important scientific terminology. Various scientific terms such as chirality, enantiomers and their biological significance, chiral compounds, chiral chromatographic separation approaches, chiral selector (for TLC), CSPs, CDRs and their applications have been discussed in brief. The CDRs have been classified in different categories depending upon the nature of functional group present in anatytes. Secoiid chapter presents the literature survey of chosen CDRs, CSPs and analytes. Third chapter deals with the description of the common experimental procedures used for present studies. It includes materials, instrumentation, preparation of stock solutions, extraction of active pharmaceutical ingredient from formulations. The details with respect to methods for synthesis of CDRs along with their characterization data, synthesis of diastereomers followed by separation using reversed phase-HPLC are described in subsequent chapters. In all, 17 CDRs were synthesized using CC (cyanuric chloride; trichloro-s-triazine) and (S)-Naproxen as starting materials. In ten of these reagents, (the DCT, dichloro-s-triazine) enantiomerically pure amines (viz., (R)-(+)-naphthy lethyl amine and (S)-(+)- I -benzyl-3-aminopyrrolidine, amino acids (viz., L-Leu, L-Val, D-Phg, L-Ala), and amino acid amides (viz., L-Leu-NH2, L-Val-NH2, D-Phg-NH2, L-Ala-NH2) were incorporated in cyanuric chloride as chiral auxiliaries. Six MCT (monochloro-s-triazine) reagents were synthesized by incorporating L-Ala-NH2, D-Phg-NH2, L-Leu-NH2, and L-Val-NH, and (R)-(+)-NEA and (S)-(+)-BAP) as chiral auxiliaries in 6- butoxy / methoxy derivative of s-triazine. Besides, (5')-Nap based CDR, (S)-2-(6-methoxynaphthalen-2-yl)propanehydrazide (NAP-H), was synthesized by its reaction with hydrazine hydrate in presence ofdicyclohexylcarbodiimide (DCC) as coupling agent. Fourth chapter presents indirect enantioseparation of amino acids, selenomethionine and amino alcohols. It has been divided into three sections. Section A: It deals with the development of a sensitive method for separation of 19 diastereomers from a mixture of thirty and determination of D-amino acids in scalemic mixtures in the absence of pure D-enantiomers. Diastereomers of 15 DL-proteinogenic amino acids were synthesized under microwave irradiation using four new CDRs. Two enantiomerically pure amines, namely, (R)-(+)-NEA, and (S)-(+)-BAP were selected as chiral auxiliaries and introduced in CC and its 6-butoxy derivative to synthesize these CDRs. The CDRs so obtained 14 were characterized and their optical purity was ascertained. The mixture of 30 diastereomers was separated on C18 column in a single run using a linear gradient of mobile phase from 100% A (Water-MeCN, 90:10) to 100% B (Water-MeCN, 10:90), containing TFA, in 50 mm (flow rate 1.0 rnL min1 detection at 230 nm). Chromatographic conditions were optimized. UV spectra of each pair of diastereomers were captured with PDA detector. Comparison of chromatogram of the multicomponent mixture with the chromatograms of the diastereomeric pairs of individual amino acids revealed that a few of the diastereomers co-eluted. Yields and stability of diastereomers were established. The separation behaviour in terms of retention times and resolutions were compared on the basis of effect of chiral auxiliaries (i.e. aniines) and achiral substituents (i.e. chlorine or butoxy group) in the CDRs and the hydrophobic side chains of amino acids. The focus of investigation of this section was (a) to develop new CDRs with enhanced UV absorption characteristics, (b) to examine/evaluate the effect of hydrophobicity of achiral substituent in CC (on enantioseparation) and (c) to obtain lower LOD in comparison to existing literature. The developed separation method was validated in terms of accuracy, precision, linearity, recovery, LOD and LOQ. Section B: This section explores the HPLC indirect resolution of DL-selenomethionine (SeMet) using four newly synthesized CC based CDRs (two DCT and two MCT). To develop CDRs, It enantionierically pure, (R)-(+)-NEA, and (S-(+)-BAP were introduced as chiral auxiliary in CC and its 6-butoxy derivative by nucleophilic substitution of one chlorine atom in each of them. The CDRs so obtained were characterized and their optical purity was ascertained. Diastereomcrs of DL-SeMet were synthesized under MWl for 60 or 90s (at 80% power of 800 W). Diastereomers were separated on a C18 column using RP-HPLC and mixtures of MeCN with aq TFA as mobile phase. The detection was made at 230 nm using PDA detector. The separation behaviour in terms of retention times and resolutions were compared. The separation method was validated for linearity, accuracy, precision, recovery and LOD. Section C. In this section an indirect HPLC enantioresolution of eight selected 13-amino alcohols has been achieved on certain CC based CDRs. In 6-methoxy triazine and CC a Cl atom was substituted with chiral auxiliaries like L-Ala, D-Phg, L-Leu, and L-Val and their amides to obtain twelve CDRs (three sets; 2 DCT and I MCT). Using MWI and each of the twelve CDRs, diastereomers of eight amino alcohols were synthesized. The resulting diastereomers were separated by RP-HPLC using C18 column and eluting mixtures of MeCN with aq.TFA in a linear gradient (45 mm) with UV detection at 230 nm. The flow rate of the mobile phase and the concentration of TFA and organic modifier in it were varied to optimize the method, so developed, for chromatographic separation of d iastereomers. The results (in terms of resolution Rs, and retention factor k) obtained for the three sets of diastereomers were compared among themselves and among the three groups. The effects of le chiral auxiliaries (in terms of acid and amides variants), constituting CDRs, on separation were also evaluated. The elution order for the diastereomers was determined. The separation method was validated for linearity, accuracy, precision, recovery and LOD. Literature survey reveals that the CC based CDRs have been applied for the first time for enantioseparation of racemic 3- amino alcohols. Fqfth chapter deals with the synthesis of naproxen hydrazide reagent (S)-2-(6-methoxynaphthalen-2-yl)propanehydrazide (NAP-I-I) followed by its application as CDR for the enantioseparation of certain carbonyl compounds. The reagent was characterized and its chiral purity was established. It was used as a CDR for the synthesis of hydrazone diastereomers, under MWI, of four aldehydes and two ketones. The respective diastereomers were resolved by RP-HPLC using C18 column and gradient eluting mixture of MeCN with aq. TFA. The separated diastereomers were detected at 231 nm using PDA detector. The method was validated for linearity, accuracy, precision, recovery and LOD. For a series of hydrazones LOD was found to be in the range 1.62 to 1.65 prnol mU'. The synthesis of naproxen hydrazide reagent using DCC as coupling reagent and its use as chiral derivatizing reagent for RP-HPLC enantioseparation of carbonyl analytes have not been reported yet. Sixth chapter is devoted to direct resolution of enantiomers of (RS)-Ketorolac (Ket) and (RS)-Amlodipine (Ami) on two polysaccharide-based chiral columns, viz. Lux Cellulose-2 and Lux Amylose-2 containing cellulose tris-(3-chloro-4-methylphenyl) carbamate and amylose tris-(5-chloro-2-rnethylphenyl) carbamate, as the chiral materials, respectively. Enantioseparation were carried out under normal and reversed phase (NP and RP) elution mode with suitable mobile phase compositions. The results obtained from two columns were compared among themselves in regards to their ability to provide baseline resolution. The effects of the nature and concentration of alcoholic modifier and pH of mobile phase on the retention and resolution were studied. The detection was made photochemically using PDA detector for identification of enantiomers. The effect of alcoholic/ organic modifier on enantioselectivity and resolution of enantiomers was evaluated. The sequence of the eluted enantiomers was ascertained by (iv) performing optical rotation studies on polarimeter. The method was validated in terms of accuracy, precision and linearity in the range of 30-70 tg mr1 and the r2 was >0.99. Seventh chapter deals with direct TLC enantioseparations of four n-blockers, namely, (RS)-atenolol, propranolol, metoprolol and bisoprolol using azithromycin (AZM) as a chiral impregnating agent as well as chiral mobile phase additive. Impregnated thin-layer plates were prepared by spreading slurry of silica gel prepared in the solution of AZM. The enantioseparation was attempted using different binary, ternary and quaternary combinations of solvents systems containing MeCN, MeOl-1, 17120 and CHC13; triethylamine was added to adjust pH. The spots were located using iodine vapor. The developed TLC method was validated for linearity, accuracy, precision, recovery and LOD. The influences of pH, temperature and amount of chiral selector were examined on enantioseparation.