CHROMATOGRAPHIC STUDIES OF CERTAIN PHARMACEUTICALLY IMPORTANT CHIRAL COMPOUNDS

Abstract

It is an established fact that enantiomers have different biological, physiological and chemical behaviour both in natural and biological systems including the human body and the environment. Enantiomerically pure compounds are needed in different areas such as chiral synthesis, mechanistic studies, pharmaceuticals, agrochemical industries, biochemistry and forensic science. Regulatory authorities, in the US particularly, have become aware that efficacy and toxicology of enantiomers may differ from each other and from the racemic mixture and are increasingly asking for data on stereochemistry. Approximately 40% of the drugs including anti-inflammatory/ analgesic, adrenergic and antihistamines, agrochemicals and food additives developed from organic synthesis possess stereogenic centre(s) but only 12% are marketed as single enantiomer. Now a day, greater attention is focused on developing methods for stereoselective and enantioselective synthesis. Nevertheless, the resolution of racemic mixtures into their enantiomers by suitable, economic and rapid methods remains important and desirable. Thus, technical issues surrounding product development have acquired greater importance. Analytical methods for separating and determining enantiomers include crystallization, chromatographic techniques such as high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), gas-liquid chromatography (GLC), thin layer chromatography (TLC), supercritical fluid chromatography (SFC) and capillary electrophoresis (CE). Currently, there are two procedures used for chromatographic resolution of enantiomers. These are classified as direct and indirect. Direct method has several advantages over indirect method, e.g., the direct approach requires no chemical derivatization with a chiral reagent prior to chromatography. To achieve direct enantiomeric resolution by chromatography following basic approaches have been mentioned in literature • Use of a chiral stationary phase (CSPs) • Use of chiral mobile phase additives (CMPAs), and • Use of chiral selector as an impregnating reagent with the sorbent. Amino acids, (3-adrenergic blocking agents, non-steroidal anti-inflammatory agents, calcium channel blockers (e.g., verapamil), thioridazine from antipsychotic phenothiazine group were selected for present studies focused on enantiomeric resolution because of their wide application in the field of chemistry, medicine, food science, forensic science and toxicology etc and easier availability. Besides, certain analytical studies on ACE-inhibitors were also carried out. The present thesis comprises of five chapters. The first chapter is introductory one dealing with historical development, theory and mechanism, nature of the adsorbent, advantages and application of TLC, theory and mechanism of HPLC and GC. The literature related to the class of compounds chosen has been cited in subsequent chapters. Second chapter deals with description of the common experimental methods used for present studies. It includes materials, equipment, preparation of TLC plates, development and detection of chromatogram and polarimetric experiments. The third chapter deals with enantiomeric separation of a few important pharmaceuticals. Various methods used and explanations provided for the resolution of a variety of compounds into their enantiomers has been briefly discussed. The chapter is divided into four sections Section A: Ligand-exchange TLC resolution of enantiomers of some racemic P-adrenergic blocking agents, namely, (±)-propranolol, (±)-metoprolol, and (±)- atenolol is described. Silica-gel plates were impregnated with optically pure Cu(II)- L-arginine complex (1:2 mmol) as the chiral complex for ligand exchange. Different combinations of acetonitrile-methanol-water system were tried and the successful solvent system resolving these compounds was worked out. Spots were detected using iodine vapour and the detection limits were established. Effect of concentration of the impregnating reagent, pH and temperature on resolution was systematically studied. 11 Section B presents application of macrocyclic antibiotic, vancomycin hydrochloride, as a chiral impregnating reagent for TLC separation of enantiomers of verapamil on thin layer silica gel plates. Mobile phase consisting of acetonitrile-methanol-water (15:2.5:2.5v/v) was found to be successful. The spots were detected with iodine vapours and the detection limit was found to be 0.147 ug. The effect of concentration of the chiral selector, temperature and pH on resolution has been studied. Section Cpresents resolution of (±)-ibuprofen using (-)-brucine as a chiral selector by thin layer chromatography using acetonitrile-methanol (5:1, v/v) as the solvent system. Spots were located in iodine chamber. The detection limit was worked out and was found to be 4.9 ug. The effect of concentration of the chiral selector, temperature and pH on resolution has been studied. Section D incorporates HPLC resolution of thioridazine enantiomers from pharmaceutical dosage form using cyclodextrin based chiral stationary phase. Thioridazine belongs to the antipsychotic phenothiazine group. It was isolated from commercial formulations and was purified using preparative TLC. The purity was ascertained by RP-HPLC. The optimum conditions of resolution were established by systematically studying the effect of organic modifier, concentration of buffer, pH and flow rate of mobile phase. The detection limit was found to be 10 ug (5 jig of each enantiomer). The enantiomeric purity of each of the resolved isomers was verified by optical rotation. Fourth chapter deals with enantiomeric resolution of amino acids. The chapter is further divided into two sections. Section A: Diastereomeric derivatives of 12 DL-amino acids were prepared with 1- fluoro-2, 4-dinitro phenyl-5-L-valine amide which is a chiral variant of Marfey's reagent. These were spotted on TLC plates. Diastereomers were separated most effectively with solvent system consisting of phenol-water (3:1, v/v) providing an indirect resolution of enantiomers . Section B presents GC/MS determination of L- and D-amino acids in fortified wines. Amino acids were isolated from fifteen fortified wines using Dowex 50Wx8 cation exchanger and converted into volatile N-(0)-perfluoroacyl alkyl esters and in analyzed on a chiral capillary column (ChiralSil®-L-Val) together with gas chromatography with flame ionization detection and selected ion monitoring mass spectrometry. The relative quantity of D-amino acid with respect to the corresponding L-enantiomer was determined in fortified wines. The Fifth chapter describes determination and separation of certain angiotensin converting enzyme inhibitors. Five antihypertensive agents namely amlodipine besylate, enalapril maleate, ramipril, lisinopril and benazepril hydrochloride were extracted, purified and crystallized from commercial formulations. These were characterized by melting point, Xmax and IR. The percentage recovery by extraction process was in the range 94-96 %. Four combinations of commercial formulations viz. amlodipine +ramipril, amlodipine + enalapril, amlodipine +benazepril and amlodipine +lisinopril were separated by HPLC and thin layer chromatography using different compositions of CHC13- CH3OH-CH3COOH as mobile phase. Detection was by UV at 226 nm in HPLC, and by iodine vapors in TLC. Quantitative determination was carried out using TLC supplemented with UV spectrophotometry. Recovery was in the range of 91- 93 °A0. Thus the experimental conditions developed and presented in this thesis represent simple methods for enantiomeric resolution of a variety of compounds using both direct and indirect approaches. In the indirect approach, Marfey's reagent was used for derivatization on one hand and (-)-brucine was used on the other hand. In the latter case it was ionic salt that was applied to TLC while in the former it was acovalently linked diastereomer. Methods involving TLC, HPLC and GC-MS have been developed. The methods are sensitive, reliable, simple and reproducible with very low detection limits. These compounds are expected to be beneficial in clinical chemistry, biochemistry and in various industries for quality control.