Development and validation of HPLC methods for the enantioselective analysis of bambuterol and albuterol.

Suitable HPLC methods for the direct separation of bambuterol and albuterol enantiomers were developed. The enantioseparation was tested on numerous commercial chiral HPLC columns. For bambuterol the most convenient separation was determined on amylose Chiralpak AD column, and for albuterol on vancomycine Chirobiotic V column. The mobile phase compositions were systematically studied to obtain the optimal chromatographic methods. Validation of methods in selected conditions shows that the chosen methods are selective and precise with linear response of detector for both pairs of enantiomers.

Mechanism of chiral recognition in the enantioseparation of 2-aryloxypropionic acids on new brush-type chiral stationary phases.

New brush-type chiral stationary phases (CSP I-IV) comprising N-3,5,6-trichloro-2,4-dicyanophenyl-L-alpha-amino acids (1-4) were prepared by binding of chiral selectors 1-4 to gamma-aminopropyl silica gel. To check the role of excess free aminopropyl groups, CSP V was prepared by binding N-3,5,6-trichloro-2,4-dicyanophenyl-L-alanyl-(3-triethoxysilyl)propylamide to unmodified silica gel. The best separation of racemic 2-aryloxypropionic acids (TR-1-13) was obtained with CSP I; the -(-)-S enantiomer were regularly eluted first, as determined by a CD detector. The mechanism of chiral recognition implies a synergistic interaction of carboxylic acid analyte with the chiral selector and achiral free gamma-aminopropyl units on silica. In fact, CSP V, which is lacking an achiral aminopropyl spacer, shows a lower separation ability for 2-aryloxypropionic acids, but a similar enantioselective discrimination of esters TR-19-20, in comparison with CSP I. CSP I-IV retain unaltered separation ability after a few months of continuous work using a large number of various mobile phases.

Solid-phase synthesis of chiral stationary phases based on 2,4,5,6-tetrachloro-1,3-dicyanobenzene derivatives spaced from N-3,5-dinitrobenzoyl alpha-amino acids: comparative study of their resolution efficacy.

Two new chiral stationary phases, 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-D-phenylglycinyl) aminoethyl]aminophen-1-yl] aminopropyl silica (CSP-1) and 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-L-leucinyl) aminoethyl] aminophen-1-yl] aminopropyl silica (CSP-2), were prepared by solid-phase synthesis. They comprise chiral unit, 3,5-dinitrobenzoyl derivative of the amino acid, D-PhGly or L-Leu, bound via spacer 1,2-diaminoethane to 2,4-positions of the persubstituted benzene ring, derived from compound 1, and possess pseudo-C2 symmetry. Preparation of model compounds 6 and 7 confirmed the structure of chiral selectors, which comprise pi-donor persubsituted aromatic ring and two strong pi-acceptor 3,5-dinitrobenzoyl amido units. CD spectra of model selectors 6 and 7, run in DMSO above 250 nm, exhibit negative exciton coupling (EC) between pi-acceptor and pi-donor chromophores, C(1) symmetric model compound 8 exhibited much weaker EC and 9, devoid of pi-donor unit, does not exhibit any significant CD. Combined pi-donor and pi-acceptor properties enable the new CSPs to separate a broad range of racemates. The columns with CSP-1 and CSP-2 were tested for the separation of 22 racemates by HPLC with two different mobile phase systems and the results are compared with those obtained by using a structurally related commercial column.

Preparation and evaluation of chiral stationary phases based on N, N-2,4-(or 4,6)-disubstituted 4,5-(or 2,5)-dichloro-1, 3-dicyanobenzene.

Regioselective functionalization of 2,4,5,6-tetrachloro-1, 3-dicyanobenzene (TCDCB) by nucleophilic substitution of the chlorine at C(4) with L-Ala, L-Phe or L-Pro, followed by amide-bond formation to lipophilic amines containing strong pi-donor group, and by final introduction of the spacer 3-aminopropyltriethoxysilyl (APTES), provided a number of new brush-type chiral selectors in the form of 1-2:1 mixture of 2,4 and 4,6-di(alkyl)amino regioisomers (8/9, 10/11, 12/13, 14/15, 20/21, 23/24). Linking these to silica gel (Nucleosil 100-5) gave new chiral stationary phases for HPLC columns (CSP I-CSP VI). Being strong pi-basic selectors, most of these columns exhibited good resolution properties for pi-acid test racemates (TR 1-TR 9), specifically rac 3, 5-dinitrobenzoyl-alpha-amino acid isopropyl-esters (DNB-AA). CSP V [1,3-dicyano-2,5(5,6)-dichloro-6(2)-(gamma'-silica bound propylamino)-4-N-¿[N-butyl]-N'-[(1R)-cyclohexylethyl]-N'-[napht hylmet hyl]acetamido¿-aminobenzene] and particularly the dipeptide-containing CSP VI [2,5(5,6)-dichloro-6(2)-(gamma'-silica bound propylamino)-4-N-(3', 5'-dimethylanilido)-L-alanyl-L-prolyl-aminobenzene] proved to have the highest efficiency, comparable with the best commercial brush-type columns with pi-donor properties. Further evidence revealed that multiple hydrogen bonding via the amide group in the chiral environment and pi-pi interaction play a major role in chiral recognition, whereas steric perturbations via nonbonding VDW interactions contribute substantially only to the resolution of CSP III [2,5(5,6)-dichloro-6(2)-(gamma'-silica bound propylamino)-4-N-(cyclohexylamido)-L-alanyl-aminobenzene]. This contribution is minor for the other CSPs.

Novel chiral stationary phases comprising 2,4-(or 2,6)-diamino-5,6-(or 2,5)-dichlorobenzene-1,3-dicarbonitrile and 1-acyl (1R,2R)-diaminocyclohexane.

Novel chiral selectors 3-5 were prepared by regioselective nucleophilic substitution of 2,4,5,6-tetrachlorobenzene-1,3-dicarbonitrile (TCBDC, 1) at C(4) by (1R,2R)-trans-diaminocyclohexane, followed by acylation of the intermediary 2 with carboxylic acids containing pi-acid or pi-basic unit. On substitution of the second chlorine atom by the spacer 3-aminopropyltriethoxysilane (APTES), a 1:1 mixture of regioisomers of N-(([3, 6-dichloro-2,4-dicyano-5-(4,4,4-triethoxy-4-silabutyl)-amino]phenyl)amino) cyclo-hexylcarboxamides and N-(5,6-dichloro-2,4-dicyano-3-(4,4,4-triethoxy-4-silabutyl)-amino]pheny) amino)cyclohexylcarboxamides (6/7, 8/9, 10/11) was obtained. Their covalent binding to Nucleosil 100-5 provided three new chiral stationary phases (CSP-1-CSP-3). NMR spectra of model compounds 12-14 and MM2 calculations on model compounds 15,16 revealed pi-pi interactions between persubstituted benzene ring and second aromatic ring. The results of the evaluation of new CSPs in the separation of 23 test racemates by HPLC are reported. CSP-2 and CSP-3, that have lower conformational freedom than CSP-1, allow for better separation. In particular, good results are obtained in the separation of some 1,4-benzodiazepines and open-chain aromatic amides by CSP-2 and CSP-3.

New chiral stationary phases based on (R)-1-naphthylethylamine bound to 2,4,5,6-tetrachloro-1,3-dicyanobenzene

Chiral functionalization of 2,4,5,6-tetrachloro-1,3-dicyanobenzene (1) by regioselective nucleophilic substitution of one or two chlorine atoms by optically pure (R)-(+)-1-naphthylethylamine (NEA), or by a glycine unit as a spacer to (R)-NEA, enables the preparation of brush-type chiral selectors (2, 3, 9, 13). By the introduction of the 3-aminopropyltriethoxysilyl (APTES) group, reactive intermediates 4a/b, 5, 10a/b, and 14a/b are obtained (a/b indicate a mixture of regioisomers with APTES in 6- and 2-position). Binding of these to silica gel afforded four novel chiral stationary phases (CSPs) 6, 7, 15, and 16. HPLC columns containing CSPs with (R)-NEA directly linked to polysubstituted aromatic ring (6, 7) are not very effective in resolution of most of the 23 racemic analytes, whereas the columns with distant pi-basic subunits (15, 16) exhibited higher resolving efficacy, in particular towards the isopropyl esters of racemic N-3,5-dinitrobenzoyl-alpha-amino acids. Effective resolution of test racemates reveals the importance of the presence of the hydrogen bond donor amido group and the distance between the persubstituted benzene ring in 1 and the pi-basic naphthalene ring of (R)-NEA. Copyright 1999 Wiley-Liss, Inc.