CHROMATOGRAPHIC CHIRAL RESOLUTION OF PHARMACEUTICAL AND BIOLOGICAL COMPOUNDS

Abstract

Chirality has emerged as a key issue in drug design, discovery and development as stereoisomer discrimination plays a significant role in many pharmacological events. It is an important factor in drug efficacy. Dosage concentration ofa racemic drug is an illusion to the patients as normally 50% drug is active while the other 50% is inactive or may be toxic. About 56% of the drugs currently in use are chiral compounds, and about 88% of these chiral synthetic drugs are administered as racemates. For enantiomeric separation on analytical scale direct and indirect separations are achieved using chromatographic methods. Indirect method is based on the use of chiral derivatization reagents (CDRs) to form diastereomeric derivatives which can be separated using an achiral stationary phase. Majority of therapeutics such as anti-inflammatory/analgesic, adrenergic and antihistamines drugs, agrochemicals and food additives are marketed as racemic mixtures. Now a days, greater attention is focused on developing methods for stereoselective and enantioselective synthesis. Hence chiral analytical methods remain important in analysis of enantiomeric purity of drugs during their different steps of synthesis, isolation from natural sources, and establishment of specifications for drug marketing as well as monitoring stereospecific fate of drugs. The presence of suitable functional groups in the molecule is a precondition of a successful derivatization. In the direct approach, either a chiral mobile phase additive (CMPA) or a chiral stationary phase (CSP) is used or the chiral selector is immobilized/ impregnated with the stationary phase. In view of the importance of enantiomeric analysis and application of chromatographic technique, studies have been carried out on enantiomeric resolution of certain compounds belonging to important pharmaceutical categories such as anesthetic, /2-blockers, /32-agonist, penicillamine, angiotensin enzyme inhibitor, and serotonin reuptake inhibitor. The enantiomeric separation has been achieved using different chiral selectors and CDRs. The first chapter is an introduction to chirality of pharmaceuticals, chiral chromatographic approaches, selection criteria of CDRs and their applications. The CDRs have been classified in different categories depending upon the nature of functional group present in analytes. The literature related to the class of compounds chosen has been cited in subsequent chapters. Second chapter deals with the description of the common experimental methods used for present studies particularly pertaining to synthesis and chromatographic (both TLC and HPLC) conditions. It includes materials, instrumentation including HPLC. The characterization data for the new CDRs is described, along with general protocols adopted for the synthesis of MR, its structural variants and the diastereomers of different compounds. CDRs synthesized are as follows: Marfey's reagent and its six structural variants, namely, FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2, FDNP-LPro- NH2, FDNP-L-Met-NH2 and FDNP-D-Phg-NH2 (CDRs-1 to 7) were synthesized using six chiral auxiliaries L-Phe-NH2, L-Val-NH2, L-Leu-NH2, L-Pro-NH2, L-Met-NH2 and D-Phg-NH2 and characterized. Chiral monochloro-s-triazines (MCTs) were synthesized by the nucleophilic displacement of chlorine atoms. One of the CI atoms was replaced by piperidine and the second CI atom in the 6-piperidinyl derivative was replaced with different amino acid amides (namely, L-Leu-NH2, L-Phe-NH2, and D-Phg-NH2) and amino acids (L-Leu and L-Ala). The present study involving the attachment of piperidine in cyanuric chloride (CC) to prepare a new CDR has been the first report in literature. Five chiral monochloro-striazines (MCTs) (CDR-8 to 12) were synthesized (namely, JV-(4-Chloro-6-piperidinyl- [l,3,5]-triazine-2-yl)-L-leucinamide, JV-(4-Chloro-6-piperidinyl-[l,3,5]-triazine-2-yl)-Lphenylalaninamide, iV-(4-Chloro-6-piperidinyl-[l,3,5]-triazine-2-yl)-D-phenylglycinamide, A^-(4-Chloro-6-piperidinyl-[l,3,5]-triazine-2-yl)-L-leucine, and N-(4-Chloro-6- piperidinyl-[l,3,5]-triazine-2-yl)-L-alanine). Third chapter presents the literature survey for the selected CDRs and analytes. Fourth chapter presents direct enantioresolution of /^-blockers and one fcantagonist. It has been divided into two sections. Section A: Direct enantioresolution by normal phase TLC using ion exchange, inclusion complexation, and ligandexchange approaches. (i) L-aspartic acid and L-glutamic acid as chiral selectors: Direct resolution of six racemic /^-blockers into their enantiomers wasachieved by normal phase TLC on silica gel plates impregnated with optically pure L-aspartic acid and L-glutamic acid as chiral selectors. (ii) Vancomycin as chiral selector: Direct resolution of four racemic beta blockers into their enantiomers was achieved by normal phase TLC on silica gel plates using pure vancomycin as a chiral impregnating agent as well as CMPA. (iii) Cu(II) complexes of L-tartaric acid, L-threonine, L-serine, D-penicillamine as chiral impregnating agents: Silica plates were impregnated with Cu(II) complexes of L-tartaric acid, L-threonine, L-serine, and D-penicillamine, for resolving four /?- blockers and one /22-agonist into their enantiomers. The Cu (II)-L-chiral selector complex was used to prepare impregnated silica gel plates. In all the cases, the influence of pH, temperature and concentration of chiral selector was studied. Detection of spots was done using iodine vapors. The TLC method was validated for linearity, and limit of detection (LOD). Different solvent systems were worked out to resolve the enantiomers. Section B: Indirect enantioresolution of /^-blockers (metoprolol, carvedilol, atenolol, propranolol) and /22-agonists (salbutamol and terbutaline) by preparing diastereomers with Marfey's reagent, its variants, (S)-N-(4- Nitrophenoxycarbonyl)phenylalaninemethoxyethyl ester {(5)-NIFE} reagent, and monochloro-.s-triazine reagent was achieved. Methods were validated for limit of detection, limit of quantification, linearity, accuracy, precision, and recovery. Both TLC and HPLC methodologies were used. in (i) Chiral separation of diastereomers prepared with MR and its variants: These were used for derivatization of metoprolol, carvedilol and terbutaline to form diastereomers. The diastereomers of metoprolol and carvedilol were separated most effectively by use of mobile phases containing acetonitrile and triethylammonium phosphate buffer by RP-TLC. The separation efficiency of the structural variants was compared with that of MR. Diastereomers of (i?,5)-terbutaline were resolved by RPHPLC using acetonitrile and 1%aq. TFA solution with gradient mobile phase. (ii) HPLC separation of diastereomers prepared with (5)-NIFE: Reversed-phase high-performance liquid chromatographic resolution of three racemic /^-blockers (metoprolol, atenolol, propranolol) and one /E-agonist (salbutamol) has been achieved using (5)-Ar-(4-nitrophenoxycarbonyl)phenylalaninemethoxyethyl ester [(5)-NIFE] as the CDR. A linear gradient of mixtures of 0.1% aq. TFA and 0.1% TFA (in MeOH) was found successful. The detection was made at 205 nm. Microwave irradiation was made to synthesize the diastereomers and reaction conditions were optimized and comparedwith the method of using conventional heating. (iii) HPLC separation of diastereomers prepared with cyanuric chloride based reagent: The diastereomers of /^-blockers (atenolol, propranolol and metoprolol) and (Jlagonist (salbutamol) were prepared with CDR-8 and separated on a reversed-phase Ci8 column using mixtures of acetonitrile and aqueous-trifluoroacetic acid (TFA). Conditions for derivatization kinetics were optimized with MCT reagent with respect to the effects of pH, reagent excess, temperature and reaction time on derivatization yield. Mechanism of separation of diastereomers has also been proposed. Fifth chapter deals with direct and indirect enantioresolution of penicillamine. Direct resolution of racemic PenA into its enantiomers was achieved by normal phase TLC on silica gel plates using optically pure L-tartaric acid as CSP as well as CMPA and also by (i?)-mandelic acid as CSP. Different solvent systems were worked out to resolve the enantiomers. Spots were detected using iodine vapors. The TLC method was validated for linearity, and limit of detection (LOD). The influence of pH, temperature and concentration of chiral selector was studied. An HPLC method was iv established for enantioseparation of DL-PenA using (5>NIFE reagent as the CDR. The method was validated for linearity, repeatability, and LOD. Sixth chapter deals with enantioresolution of fluoxetine in pharmaceutical formulations by both direct and indirect methods using HPLC and TLC. L-tartaric acid has been used as a mobile phase additive in TLC and the enantiomers were separated and isolated and were used to determine the elution order in HPLC. (^?,5)-flouxetine was derivatized with (5)-NIFE, Marfey's reagent and l-fluoro-2,4-dinitrophenyl-Lmethionine amide (FDNP-L-Met-NH2). The diastereomers were separated using RPHPLC. The effect of flow rate, and TFA concentration on resolution was studied. The diastereomers obtained by derivatization with FDNP-L-Met-NH2 were also separated by RP-TLC. Seventh chapter deals with enantioresolution of DL-amino acids, amino alcohols, /2-blockers and a /2-agonist via synthesis of diastereomers prepared with (S)- NIFE as the CDR using reversed-phase high-performance liquid-chromatographic method. It is divided in two sections: Section A: Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, and PenA) was done using the reagent (5)-NIFE by RP-HPLC. The diastereomeric derivatives were analyzed under reversed phase conditions using linear gradient of mixtures of 0.1% aq. TFA and 0.1% TFA in MeOH. Sharp peaks were obtained under these chromatographic conditions. The detection was at 205 nm. Method validation was also done. Section B: CDRs 8 to 12 were used for the synthesis of distereomers of /^-blockers (metoprolol, atenolol, and propranolol), /2-agonist (salbutamol), penicillamine, 18 protein and 08 non-protein amino acids. The resultant diastereomers were separated on a reversed-phase C18 column using mixtures of acetonitrile and aqueous-trifluoroacetic acid (TFA). The reaction conditions were optimized for derivatization kinetics with CDR-1 with respect to the effects of pH, reagent excess, temperature and reaction time on derivatization yield. HPLC method was also validated for limit of detection, limit of quantification, linearity, and recovery. Effects of structural modifications in reagents on chromatographic properties were investigated. Separation mechanism of diastereomers has been proposed. Eighth chapter presents direct TLC enantioresolution of ketamine and Hsinopril belonging to the class of anesthetic and angiotensin enzyme inhibitor, respectively. Direct resolution of racemic ketamine and Hsinopril into their enantiomers was achieved by normal phase TLC on silica gel plates impregnated with optically pure L-tartaric acid and (i?)-mandelic acid as chiral impregnating agent and/or as CMPA. Different solvent systems were worked out to resolve the enantiomers. Spots were detected using iodine vapor. The TLC method was validated for linearity, and limit of detection (LOD). The influence of pH, temperature and concentration of chiral selectors was studied.