Immobilized halogenophenylcarbamate derivatives of cellulose as novel stationary phases for enantioselective drug analysis.

Three different halogeno-phenylcarbamate derivatives of cellulose have been prepared and coated on silica gel. The coated materials have been immobilized and their chiral recognition ability as chiral stationary phase (CSP) has been evaluated with a set of reference racemates, including several drugs such as lormetazepam, glutethimide, and warfarin, using various mobile phase mixtures. The novel phases were found to exhibit unique enantioselective properties compared with more established polysaccharide-based CSPs. A good resolution of all racemates could be successfully achieved on at least one of the immobilized CSPs. Moreover, it has been pointed out that selectivity may considerably vary with the composition of the mobile phase.

Enantioselective chromatography as a powerful alternative for the preparation of drug enantiomers.

The preparative separation of enantiomers by chromatography on chiral stationary phases (CSPs) has been recognized as being a useful alternative to the more conventional approaches such as enantioselective synthesis and enzymatically catalyzed transformations. The possible contribution of enantioselective chromatography with respect to the preparation of enantiomerically pure compounds is reviewed in the context of the competitive approaches and depending on the application scale, with a special emphasis on the recent progresses achieved in this particular field of separation.

Chiral resolution, pharmacological characterization, and receptor docking of the noncompetitive mGlu1 receptor antagonist (+/-)-2-hydroxyimino- 1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester.

Racemic CPCCOEt ((1aRS,7aRS)-2-hydroxyimino-1a, 2-dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic acid ethyl ester, (+/-)-1) derivatives have been shown to be subtype-selective metabotropic glutamate (mGlu) 1 receptor antagonists (Annoura et al. Bioorg. Med. Chem. Lett. 1996, 6, 763-766). The optical isomers of (+/-)-1 have been separated by chromatography on a chiral stationary phase. The absolute configuration at the C-1a and C-7a positions was determined using X-ray crystallography of an amide derivative with the methyl ester of L-phenylalanine (L-PheOMe) ((+)-6). In a phosphoinositol (PI) turnover assay at the cloned human mGlu1b receptor, (-)-1 and the new amide derivatives (-)-5 and (-)-6, all of which have (1aS,7aS)-stereochemistry on the chromane ring system, showed IC(50) values of 1.5, 0.43, and 0.93 microM, respectively. In contrast, (+)-1 and the new amide derivatives (+)-5 and (+)-6were found to be inactive up to a concentration of 30 microM indicating a selectivity for the (-)-enantiomers of at least 70-fold. In a previous study (Litschig et al. Mol. Pharmacol. 1999, 55, 453-461) we demonstrated using site-directed mutagenesis that the interaction site of (+/-)-1 is located in the transmembrane (TM) domain of hmGlu1b. To suggest a plausible binding mode of (-)-1, we have built a molecular mechanics model of the putative seven TM domain of hmGlu1 based on the alpha-carbon template of the TM helices of rhodopsin. A receptor docking hypothesis suggests that the OH of T815 (TMVII) comes in close contact with the oxime OH of (-)-1 and (-)-5, whereas no such close interactions could be demonstrated by docking of (+)-1.

(+)-4-phosphonophenylglycine (PPG) a new group III selective metabotropic glutamate receptor agonist.

A new synthesis of (R,S)-PPG (4-phosphonophenylglycine) and the separation of the protected enantiomers leading after deprotection to (+)- and (-)-PPG are described. Pharmacological characterization at the group III metabotropic glutamate receptors hmGluR4a and hmGluR7b revealed (+)-PPG as the active enantiomer.

Separation of enantiomers by packed capillary electrochromatography on a cellulose-based stationary phase.

Separation of enantiomers was performed by applying packed capillary electrochromatography (CEC). Fused-silica capillaries of different lengths with an inner diameter of 100 microm were packed with a cellulose derivative immobilized onto macroporous silica gel. Parameters such as content of modifier in the mobile phase, concentration and pH of the buffer were varied for a set of test capillaries to determine their influence on enantioselectivity. In packed CEC the highest influence on resolution of the test racemates was found by changing the acetonitrile content, while variation of the buffer concentration mostly affects the electroosmotic velocity. The performance of packed CEC and nano-LC was also compared. Packed CEC showed much better column efficiency and enantioselectivity under similar flow/electroosmotic velocity.

Chiral inversion and hydrolysis of thalidomide: mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites.

The chiral inversion and hydrolysis of thalidomide and the catalysis by bases and human serum albumin were investigated by using a stereoselective HPLC assay. Chiral inversion was catalyzed by albumin, hydroxyl ions, phosphate, and amino acids. Basic amino acids (Arg and Lys) had a superior potency in catalyzing chiral inversion compared to acid and neutral ones. The chiral inversion of thalidomide is thus subject to specific and general base catalysis, and it is suggested that the ability of HSA to catalyze the reaction is due to the basic groups of the amino acids Arg and Lys and not to a single catalytic site on the macromolecule. The hydrolysis of thalidomide was also base-catalyzed. However, albumin had no effect on hydrolysis, and there was no difference between the catalytic potencies of acidic, neutral, and basic amino acids. This may be explained by different reaction mechanisms of the chiral inversion and hydrolysis of thalidomide. Chiral inversion is deduced to occur by electrophilic substitution involving specific and general base catalysis, whereas hydrolysis is thought to occur by nucleophilic substitution involving specific and general base as well as nucleophilic catalysis. As nucleophilic attack is sensitive to steric properties of the catalyst, steric hindrance might be the reason albumin is not able to catalyze hydrolysis. 1H NMR experiments revealed that the three teratogenic metabolites of thalidomide, in sharp contrast to the drug itself, had complete chiral stability. This leads to the speculation that, were some enantioselectivity to exist in the teratogenicity of thalidomide, it could result from fast hydrolysis to chirally stable teratogenic metabolites.

Simulated moving bed chromatographic resolution of a chiral antitussive.

The behavior of a laboratory simulated moving bed (SMB) unit for continuous chromatographic separation of enantiomers has been considered. This was applied to the resolution of a chiral antitussive agent, guaifenesin, on Chiralcel OD, during an experimental campaign involving nineteen runs. The application of recently developed criteria for the design and optimization of SMB units allows us to understand and rationalize the experimental results, as well as to indicate how to optimize the separation performances. A three-step procedure to determine the adsorption isotherms needed to apply these criteria is proposed; it is reliable and may be applied also where pure components are not available.

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester.

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 micron silica-bonded, alpha 1-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1-4% (r > 0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50 degrees C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r > 0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.

Dextrin sulfopropyl ether: a novel anionic chiral buffer additive for enantiomer separation by electrokinetic chromatography.

Dextrin 10 sulfopropyl ether (DSPE) was prepared and characterized by matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) using a recently developed matrix. The anionic compound was used as a novel chiral buffer additive for enantiomer separation by capillary electrophoresis, thereby changing into the elektrokinetic chromatography (EKC) mode. DSPE was systematically evaluated as a chiral selector and was compared to the respective nonderivatized maltodextrin. DSPE showed an increased separation power for cationic racemic solutes. Although not quite as versatile and powerful as cyclodextrins, the inexpensive dextrin 10 and its derivative DSPE showed remarkable enantioselectivity in some cases.

Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds.

The enantiomers of the potent nonsteroidal inhibitor of aromatase fadrozole hydrochloride 3 have been separated and their absolute configuration determined by X-ray crystallography. On the basis of a molecular modeling comparison of the active enantiomer 4 and one of the most potent steroidal inhibitors reported to date, (19R)-10-thiiranylestr-4-ene-3,17-dione, 7, a model describing the relative binding modes of the azole-type and steroidal inhibitors of aromatase at the active site of the enzyme is proposed. It is suggested that the cyanophenyl moiety present in the most active azole inhibitors partially mimics the steroid backbone of the natural substrate for aromatase, androst-4-ene-3,17-dione, 1. The synthesis and biological testing of novel analogues of 3 used to define the accessible and nonaccessible volumes to ligands in the model of the active site of aromatase are reported.

Separation of the enantiomers of some racemic nonsteroidal aromatase inhibitors and barbiturates by capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC) were applied to the resolution of racemic nonsteroidal antiaromatase drugs and intermediates. Successful results were obtained in both modes using alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD), or 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD) as chiral selectors. Depending on the structure of the solute, one of the cyclodextrins was generally better suited for resolution of the racemate. The basic solutes were analyzed under HPCE conditions, whereas the nonionizable compounds such as glutethimide (Doriden) were analyzed in MECC mode. For the azole-type antiaromatase Fadrozole, both HPCE and MECC modes could be used to achieve the separation of the enantiomers. The influence of experimental factors such as pH, the presence of organic modifier, temperature, the micelle concentration, and the concentration of the chiral selector is also discussed on the basis of the results obtained with some chiral barbiturates. The possibility of analyzing the enantiomers directly in plasma samples was also demonstrated.

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.

Preparative chromatographic separation of enantiomers.

The potential of the chromatographic separation of enantiomers on a preparative scale as a tool for the isolation of optically pure compounds is gaining increasing recognition. This review surveys the different chiral stationary phases (CSPs) used for preparative chromatography, emphasizing the advantages and drawbacks of each. The strategy to be followed for preparative separations is discussed and tables summarizing separations reported in the literature give an overview of practical applications. Cellulose triacetate has been used most frequently, probably because of its broad application range and its low production costs in comparison with more recently introduced CSPs. Nevertheless, the high efficiency of some of the novel CSPs is likely to contribute to the further development and expansion of the method.

Synthesis and biological activity of 2-lactonyl penems.

A series of potent antibacterial agents have been prepared. These agents are penems carrying a lactone ring in the C-2 position. Excellent activity against Gram-positive and Gram-negative organisms--except Pseudomonas aeruginosa--was found.

Isolation and stereospecific determination of the enantiomers of oxindazac by direct liquid chromatographic resolution on triacetylcellulose.

The preparation of the optically pure enantiomers of the antiphlogistic trial drug oxindazac via liquid chromatographic resolution of the corresponding tert.-butyl or benzyl ester on triacetylcellulose is described. Cleavage of the optically pure enantiomeric esters to the acids proceeds without significant racemization. The methyl ester of oxindazac is also completely resolved on the same chiral phase. Whereas oxindazac racemizes easily upon derivatization to diastereomers, no racemization is observed upon methylation to the corresponding methyl ester with diazomethane. An inverse isotope dilution method has been developed to determine both enantiomers of the drug in biological fluids after administration of 14C-labelled oxindazac. The enantiomers are converted into their methyl esters and separated on triacetylcellulose. Quantitation is performed by on-line UV detection at 290 nm and off-line radiometry. In the analysis of plasma samples, endogenous compounds do not interfere. The recoveries of [14C]oxindazac from water, rat and human plasma were 99.6 +/- 1.8% for the (+/- and 96.0 +/- 1.4% for the (-)-enantiomer. The plasma concentrations and urinary excretion of the two enantiomers were determined in a human volunteer who had received 200 mg of racemic 14C-labelled oxindazac.