ENANTIOSEPARATION OF CERTAIN PHARMACEUTICAL COMPOUNDS BY LIQUID CHROMATOGRAPHY

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. Usually the desired pharmacological activity resides in one enantiomer while the other enantiomer may be toxic, less active or possesses undesirable side effects. Therefore, United State Food and Drug Administration, European Committee for Proprietary Medicinal Products and other drugs regulatory agencies have restricted the marketing of racemic drugs unless the pharmacodynamics and pharmkinetics data of each of the enantiomers is made available and declared safe£) 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 liquid chromatographic enantiomeric resolution of baclofen, mexiletine, ^-blockers, a-amino acids (proteinogenic and non-proteinogenic) and certain chiral carbonyl compounds. These compounds have wide applications in the field of chemistry, biochemistry, medicine, etc. First chapter deals with preamble to present studies, a brief introduction to the applicable scientific terms, literature related to the classes of chosen chiral dcrivatizing reagents (CDRs) and analytes. Second chapter deals with the description of materials, instrumentation and methods for synthesis of CDRs along with their characterization data. Methods for synthesis of diastereomers of chosen analytes and general experimental details with respect to their HPLC separation have also been discussed. A total of 24 CDRs were synthesized using CC (cyanuric chloride; trichloro-s-triazine) and DFDNB (difluoro dinitrobenzene) as structural moieties/synthons. Of these 24 CDRs, 13 DCT (dichloro-s-triazine) reagents (CDR 1-13) were synthesized by substitution of one of the chlorine atoms in CC with L-Leu, L-Val, DPhg, L-Met, L-Ala, L-Leu-NH2, L-Val-NH2, D-Phg-NH2, L-Met-NH2, L-Ala-NH2, (S)- (-)-a,4-dimethylbenzylamine, (-)-cw-myrtanylamine and (i?)-(-)-l-cyclohexylethylamine as chiral auxiliaries. Five MCT (monochloro-s-triazine) reagents (CDR 14-18) were synthesized by substitution of one of the chlorine atoms in 6-methoxy derivative (i) of CC with L-Leu-NH2, L-Val-NH2, D-Phg-NH2, L-Met-NH2 and L-Ala-NH2 as chiral auxiliaries. The remaining MCT reagent (CDR 19) was synthesized by substitution of two chlorine atoms with L-Val-NH2 and L-Phe-NH2 in CC. Literature reveals that this is the first report on exploring amino acids and amines as chiral auxiliaries in CC for synthesis of new DCT reagents. A new series of CDRs consisting of five hydrazino dinitrophenyl (HDNP)- amino acid reagents (CDR 20-24; HDNP-L-Leu, HDNP-L-Val, HDNP-L-Phe, HDNPL- Ala and HDNP-D-Phg) was prepared by substitution of the fluorine atom of corresponding fluoro-dinitrophenyl (FDNP)-amino acid with hydrazine hydrate. The FDNP-amino acids, in turn, were prepared by substituting one of the fluorine atoms in DFDNB moiety with amino acids (namely, L-Leu, L-Val, L-Phe L-Ala, D-Phg). Third chapter deals with enantioresolution of (i?,5)-mexilitine via diastereomerization with nineteen CC based CDRs. The diastereomers were synthesized using microwave irradiation (MWI) for 60 s at 85% power using DCT reagents and 90 s at 85% power using MCT reagents. These were resolved by reversedphase (RP)-HPLC using C\» column and gradient eluting mixtures of MeOH/MeCN and aq TFA with UV detection at 230 nm. Experimental conditions were optimized for synthesis and chromatographic separation of diastereomers. Separation efficiencies of different CDRs were compared on the basis of effect of chiral auxiliaries (i.e., amino acids or amino acid amides or amines) and achiral substituents (i.e., chlorine or methoxy group) constituting them. DCT reagents having amino acids as chiral auxiliaries were observed to be superior as compared to their counterparts having either amino acid amides or amines as chiral auxiliaries in terms of providing better resolution of diastereomers. Explanations have been offered for longer retention times and better resolution of diastereomers prepared with DCT reagents in comparison of their MCT counterparts and, for the influence of hydrophobicities of the side chains of the amino acids in the CDRs on retention times and resolution. The method was validated for limit of detection (LOD), limit of quantitation (LOQ), linearity, accuracy and precision. Literature survey shows that these 19 CDRs have been applied for the first time for enantioseparation of (R,S)-MEX. Fourth chapter presents indirect HPLC and direct TLC enantioresolution of (i?,,S)-baclofen. Indirect RP-HPLC separation of enantiomers was accomplished in the (ii) form of its diastereomers prepared with the same 19 CDRs as were used for enantioseparation of (R,S)-MEX. Optimization of chromatographic separation conditions was carried out by varying the nature and concentration of organic modifier, flow rate and TFA concentration. Among all the 19 pairs of diastereomers, the diastereomeric pair prepared with DCT reagent having L-Leu as chiral auxiliary (CDR 1) was observed to have the highest resolution with mobile phase consisting of eluent A [MeOH (100 mL) + H20 (900 mL)] and eluent B [MeOH (800 mL) + H20 (200 mL)], both eluents containing 0.10% TFA with a linear gradient of 0-100% B in 45 min. The separation behavior of diastereomers prepared with different reagents was compared. Direct TLC enantiomeric resolution of (i?,5)-baclofen along with isolation of native enantiomers was achieved using L-Lys and L-Arg as chiral impregnating reagents. Experiments were carried out to study the effects of temperature, pH and concentration of chiral selectors on enantioresolution. Literature reveals that this is the first report on direct TLC enantioresolution of (i^^-baclofen. Fifth chapter deals with enantioseparation of five ^-blockers, namely, (R,S)- atenolol, (^^-propranolol, (/?,5)-bisoprolol, (/?,5)-metoprolol and (^^-carvedilol by HPLC. A total of seventy-five pairs of diastereomers were synthesized using fifteen CC based CDRs under MWI. Separation of diastereomers was carried out on Cis column and gradient eluting mixtures of acetonitrile with aq TFA with UV detection at 230 nm. DCT reagent having the chiral auxiliary of L-Leu (CDR 1) provided best resolution for the diastereomers of (R,S)- atenolol and (R,S)- propranolol. On the other hand, DCT reagent having the chiral auxiliary of L-Val (CDR 2) provided best resolution for the diastereomers of (/?,,S)-bisoprolol, (^,S)-metoprolol and (7?,5)-carvedilol. The method was validated for LOD, LOQ, linearity, accuracy and precision. Sixth chapter deals with enantioseparation of twenty four a-amino acids. DCT reagents having L-Leu, D-Phg, L-Val, L-Met, L-Ala and L-Met-NH2 as chiral auxiliaries in CC were introduced for enantioseparation of thirteen proteinogenic amino acids. These six CDRs along with four other DCT and six MCT reagents having amino acid amides as chiral auxiliaries were used for derivatization of analytes under MWI (using 60-90 s at 85% power of 800 W). All the diastereomers were also synthesized using conventional heating and chromatographic results were compared for the two (iii) approaches. The best resolution of all the 13 analytes was observed for their diastereomers prepared with the DCT reagent having L-Leu as chiral auxiliary. Subsequently, it was further applied for derivatization of Lys, Tyr, His and Arg followed by RP-HPLC analysis of the resulting diastereomers. The same six DCT reagents along with four other DCT reagents having amino acid amides as chiral auxiliaries were also used for synthesis of diastereomers of seven non proteinogenic a-amino acids under MWI. The diastereomers were separated by RP-HPLC using gradient eluting mixtures of acetonitrile with aq TFA. The separation results are discussed in the light of (a) acid and amide groups of chiral auxiliaries of CDRs (b) electronegativities of the atoms of achiral moieties constituting CDRs and (c) hydrophobicities of side chains of amino acids constituting CDRs and analytes. Seventh chapter deals with synthesis of five HDNP-amino acids reagents (HDNP-L-Leu, HDNP-L-Val, HDNP-L-Phe, HDNP-L-Ala and HDNP-D-Phg). These five CDRs were used for synthesis of diastereomers of six racemic carbonyl compounds. The thirty diastereomers were resolved by RP-HPLC using C\% column and gradient eluting mixture of acetonitrile or methanol with triethylammonium phosphate (TEAP) buffer with UV detection at 348 nm. The novelty of the work presented in this chapter lies in (a) altering the functionality of DFDNB based CDRs for enantioseparation of analytes having functional group other than amino, particularly carbonyl (b) use of amino acids (namely, L-Leu, L-Val, L-Phe, L-Ala and D-Phg) as chiral auxiliaries for synthesis of new chiral hydrazine reagents and, (c) application of MWI for synthesis of CDRs and diastereomers.