LIQUID CHROMATOGRAPHIC ENANTIORESOLUTION OF PHARMACEUTICALLY IMPORTANT COMPOUNDS

Abstract

Chiral resolution of enantiomers is one of the emerging areas as the enantiomers of a chiral drug have different pharmacological effects as only one enantiomer of the drug often exhibits the desirable therapeutic activity, while the other shows an antagonistic function, side effects, or even toxic effects. These properties of the enantiomers have created an interest to study the pharmacological and toxicological behaviors of the individual enantiomers of drugs, pharmaceuticals, and agrochemicals. The United States Food and Drug Administration has issued guidelines to pharmaceutical and agrochemical industries to specify the enantiomeric purity of the optically active compounds prior to their marketing and hence demanded a systematic investigation of the biological behavior of their individual enantiomers and significantly encouraged the development of single enantiomer drugs. In view of these facts, the enantiomeric resolution of a variety of compounds is gaining importance continuously. There exists a multitude of methods and techniques specifically designed for enantiomeric separations, though not all methods are equally applicable for every racemic mixture. Drug development within the pharmaceutical industry focuses heavily on asymmetric synthesis, enzymatic resolution, crystallization methods, chromatographic techniques such as high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), high-performance thin layer chromatography (HPTLC), gas-liquid chromatography, and supercritical fluid chromatography, membrane processes and combinatorial chemistry. Of the possible methods for obtaining enantiomerically pure compounds, chromatographic techniques particularly HPLC is most commonly employed. Considering the biological importance of chirality and the need for chiral separation from academic, industrial, and biomedical points of view, studies have been carried out on direct and indirect enantiomeric resolution of cinacalcet, penicillamine, cysteine, homocysteine, mexiletine, omeprazole, lansoprasole, rabeprazole, pantoprazole, and amino acid analogues amino alcohols which are of great pharmaceutical and biomedical significance. First chapter provides the introduction to present studies followed by summary of present work and the brief introduction to the applicable scientific terms. Objective of present work, selection of chiral compounds for enantioseparation purpose, chiral chromatographic separation approaches, chiral stationary phases (CSPs), chiral derivatizing reagents (CDRs) and various scientific terms such as chirality and its biological importance have been discussed in brief. The literature related to the class of compounds chosen has been cited in subsequent chapters. Second chapter describes the common experimental methods used for present studies. It includes materials, instrumentation, methods for synthesis of two new chiral derivatizing reagents (CDRs) and six others. Six CDRs namely 1-Fluoro-2,4-dinitrophenyl-L-alaninamide (FDNP-L-Ala-NH2; CDR 1), 1 -Fluoro-2,4-dinitrophenyl-L-phenylalaninamide (FDNP-L-Phe-N 12; CDR2), 1-Fluoro-2,4-dinitrophenyl-L-valinamide (FDNP-L-Val-NH2; CDR3), 1-Fluoro-2,4-dinitrophenyl-L-leucinamide (FDNP-L-Leu-NH2; CDR4), 1-Fluoro-2,4-dinitrophenyl-L-methioninamide (FDNP-L-Met-N}{2; CDR5), and 1-Fluoro-2,4-dinitrophenyl-D-phenylglycinamide (FDNP-D-Phg-NH2; CDR6) were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene (DFDNB) with six amino acid amides namely L-Ala-NH2, L-Phe-NH2, L-Val-NH2, L-Leu-NH2, L-Met-NH2 and D- Phg-NH2, respectively. o new CDRs namely (S)-naproxen-benzotriazole and (S)-naproxen-benzimidazole were thesized by reaction of (S)-Nap with 1H-benzotriazole and benzimidazole, respectively, ig coupling reagent dicyclohexyl carbodiimide and 4-dimethylamino pyridine. The racterization data for the new CDRs is also described. The details with respect to synthesis of ;tereomers and separation by HPLC are described in different chapters along with results and ;ussions. Third chapter deals with enantioresolution of (R,S)-cinacalcet by using both indirect and direct approaches. For indirect approach, cinacalcet was derivatized with six chiral variants based on DFDNB (i.e. CDRs 1-6) under microwave irradiation (MWI) conditions. The derivatization conditions were optimized with respect to role of the pH of the base, effect of the CDR excess to the cinacalcet and MWI conditions. The synthesized diastereomers were resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) and ultraviolet (UV) detection at 340 nm using binary mixtures of aqueous trifluoroacetic acid and acetonitrile. Effect of flow rate and various linear gradients was also studied. However using the direct approach the enantiomers of cinacalcet were resolved on thin silica gel layers impregnated with optically pure L-His and L-Arg. Effects of the variations in the concentrations and pH of impregnating reagents and temperature were also studied. Both the resolution approaches were validated. Fourth chapter deals with enantioresolution of thiol-group containing a,-amino acids namely penicillamine, cysteine, and homocysteine by RP-HPLC. These amino acids were derivatized with (S)-naproxen-benzotriazole under MWI conditions. The derivatization conditions were optimized with respect to effect of the pH of the base, effect of the CDR excess and MWI conditions. The resultant diastereomers were resolved on a reversed phase column with gradient elution of triethylammonium phosphate (TEAP)-acetonitrile and UV detection at 231 nm. The effects of pH and concentration of TEAP buffer, organic solvent, and flow rate on RP-HPLC separation were studied. The separation mechanism was explored for the three pairs of diastereomers prepared the said CDR. The method was validated for accuracy, precision, and limit of detection. Fifth chapter explores the HPLC indirect resolution of mexiletine using (St)-(-)-(1V)- trifluoroacetyl-prolyl chloride and (15)-(-)-camphanic chloride. Kinetic resolution• method was also applied to yield a diastereomer in excess. The mole ratio of mexiletine to each of the CDRs was investigated to overcome the kinetic resolution. The synthesized diastereomers were subjected to HPLC using reversed phase column and binary composition of aqueous trifluoroacetic acid-acetonitrile as mobile phase and UV detection at 210 nm. This analytical enantioresolution method was optimized, validated, and scaled up to small-scale preparative enantioresolution method only for those resultant diastereomers, which were significantly enriched with one diastereomer, yielded due to kinetic resolution method. The separated diastereomers, which were collected on small-scale preparative enantioresolution method were hydrolyzed using hydrochloric acid and acetic acid to yield (R)-mexiletine, . the pharmacologically desired native enantiomer. Sixth chapter explores the direct HPLC enantioresolution of four anti-ulcer drugs namely omeprazole, lansoprazole, rabeprazole, and pantoprazole. The enantioresolution of these anti-ulcer drugs (i.e. chiral sulfoxides) was perfomed to investigate the enantioseparation capability of polysaccharide-based Lux cellulose-2 chiral stationary phase under normal and polar-organic phase conditions. The detection was carried out using UV detection at 285 nm. The method was validated for linearity, accuracy, precision, robustness, and limit of detection. The optimized enantioresolution method was compared for both the elution modes. The column was not stultified even after a number of bimodal elutions. The optimized method was further utilized to check the enantiomeric purity of dexrabeprazole drug. Seventh chapter deals with indirect HPLC resolution of enantiomers of eight n-amino alcohols; DL-alaninol, DL-leucinol, DL-prolinol, DL-phenylalaninol, DL-phenylglycinol, DL-valinol, DL-homophenylalaninol and DL-methioninol. These amino alcohols were derivatized with (S)-naproxen-benzimidazole under MWI conditions. The derivatization conditions were optimized with respect to effect of the pH of the base, effect of the CDR excess and MWI conditions. The resultant diastereomers were resolved on a reversed phase column with gradient elution of TEAP-acetonitrile at 231 nm. The effects of pH and concentration of TEAP buffer, organic solvent, and flow rate were studied. The separation mechanism was explained for the diastereomers prepared with the CDR. The method was validated for accuracy, precision, and limit of detection.