Stereoselective Separation Of Compounds With Two Chiral Centers By Countercurrent Chromatography

Enantiomers of compounds containing a single chiral center have similar physical and chemical properties,but they might show complete difference in pharmacologic and toxicologic activities.Compounds containing multiple chiral centers are more likely to be complictated due to increasing number of stereoisomers.Big difference in pharmacologic activities might be found between enantiomers and diastereoisomers due to the stereospecific characteristics of chemical structures,which necessitate a method for preparative stereoselective separation.However,the stereoselective separation of such type of racemate by various kinds of chromatographictechniqueisstillaverychallengingtask.Countercurrent chromatography is a kind of liquid-liquid partition chromatography using no solid support for the stationary phase without irreversible absorption involved during separation,and it has been widely used for preparative separation of active components from natural products.However,only very limited number of literatures is available,which are about chiral separations by countercurrent chromatography due to its low theoretical plates of separation column.However,in the recent years,application of countercurrent chromatography in chiral separations has been attracted much more attention since it is an ease-to-be scaled up technique.In the present work,fourβ-adrenergic blocking agents,the antidepressantdrugtrans-paroxetineanditstwoprecursors,2-[4-（2-oxocyclopentylmethyl）phenyl]propionate,a key intermediate for preparation of the nonsteroid anti-inflammatory drug loxoprofen,were stereoselectively separated by countercurrent chromatography with different chiral selectors.Three elution modes,including traditional countercurrentchromatography,pH-zone-refiningcountercurrent chromatography and recycling elution mode,were involved during the research.The main contents of this paper are as followings:（1）Fourβ-adrenergic blocking agents were stereoselectively separated by conventional countercurrent chromatography based on complexation of di-n-hexyl L-tartrate and boric acid,in which two of them containing two chiral centers.This is the first time report on successful stereoselective separation of optical isomeric compounds containing two chiral centers by countercurrent chromatography.A two-phase solvent system composed of chloroform:0.05 mol L^-11 of acetate buffer containing 0.10 mol L^-11 of boric acid（1:1,v/v）was selected by enantioselective liquid-liquid extraction,in which 0.10 mol L^-11 of di-n-hexyl L-tartrate was added in the organic phase as chiral selector.20 mg-42 mg of each racemate was stereoselectively separated by countercurrent chromatography in a single run with high purity of 96%-98%,and the recovery of each separated compound reached around 87%-93%.At the same time,a chiral stationary phase was screened for analytical stereoselective separation of the two chiral centers compounds by high performance liquid chromatography.（2）Twoβ-adrenergic blocking agents were enantioseparated by pH-zone-refining countercurrent chromatography.A two phase solvent system composed of chloroform containing 0.10 mol L^-11 of di-n-hexyl L-tartrate:0.10 mol L^-11 of boric acid aqueous solution（1:1,v/v）was selected,in which 20 mmol L^-11 triethylamine was added in the organic phase as a retainer and 2 mmol L^-11 HCl was added in the aqueous phase as an eluter.50 mg of each racemate was completely enantioseparated by pH-zone-refining countercurrent chromatography to yield each enantiomer with a purity of more than 98%,and the recovery of each separated enantiomer reached around 76%-82%.The effects of eluter and retainer on the chiral separation of pH-zone-refining countercurrent chromatography were investigated and discussed.（3）Racemictrans-paroxol,trans-N-methylparoxetineand trans-paroxetine containing two chiral centers were stereoselectively separatedbycountercurrentchromatographyusing hydroxypropyl-β-cyclodextrin as chiral selector.A two-phase solvent system composed of n-butyl acetate:0.1 mol L^-11 sodium carbonate-sodium bicarbonate buffer（1:1,v/v）at pH=9.2 was selected,in which 0.10 mol L^-1hydroxypropyl-β-cyclodextrin was added to the aqueous phase as chiral selector.20 mg of racemic trans-N-methylparoxetine and racemic trans-paroxolwerestereoselectivelyseparatedbycountercurrent chromatography in an individual run,respectively,yielding 7.1-8.3 mg of enantiomer with high purity of being 95%-98%,and the recovery of each separated isomer reached around 70%-83%.A 20 mg of racemic trans-paroxetine was stereoselectively separated by countercurrent chromatography in a recycling elution mode with a biphasic solvent system composed of n-hexane:n-butylacetate:0.1mol L^-1sodium carbonate-sodium bicarbonate buffer（9:1:10,v/v/v）at pH=9.2 was selected,in which 0.10 mol L^-11 hydroxypropyl-β-cyclodextrin was added in the aqueous phase as chiral selector,yielding 5.6 mg of enantiomer with high purity of being more than 99%.Different elution sequence and elution performance by countercurrent chromatography was found beween trans-N-methylparoxetine and trans-paroxetine,which might be caused by the different substituted group of piperidine ring on their structures.（4）Recycling countercurrent chromatography was successfully applied to resolution of 2-（4-bromomethylphenyl）propionic acid,a key synthetic intermediate for synthesis of nonsteroidal anti-inflammatory drug loxoprofen,using hydroxypropyl-β-cyclodextrin as chiral selector.The two-phase solvent system composed of n-hexane:n-butyl acetate:0.1 mol L^-11 citrate buffer solution with pH=2.4（8:2:10,v/v/v）was selected.50 mg of 2-（4-bromomethylphenyl）propionic acid was enantioseparated using preparative recycling countercurrent chromatography.The purities of both（S）-enantiomer and（R）-enantiomer were over 99.0%as determined by HPLC.Enantiomeric excess of（S）-enantiomer and（R）-enantiomer reached98.0%.Recovery for enantiomers from eluted fractions was 40.8-65.6%,yielding 16.4 mg of（S）-enantiomer and 10.2mg of（R）-enantiomer.Unfortunately,no successful enantioseparation was achieved for loxoprofen by countercurrent chromatography using the same solvent system and chiral selector,and further screening of solvent system and chiral selector were required.