Laure Hitzel

An Increased Throughput Method for the Determination of Partition Coefficients

AbstractPurpose. To present an increased throughput automated shake-flask method for the direct determination of the partition coefficients of solutes between octan-1-ol and buffer. Method. The traditional shake-flask method has been transferred onto 96-well plate technology and a robotic liquid handler has been used for sample preparation. A custom programmed Gilson auto- sampler samples the organic and aqueous phases directly from the plate, circumventing the need for any manual separation. Analyses are performed by reverse phase high performance liquid chromatography (RP-HPLC). Generic fast gradient RP-HPLC conditions are used to eliminate chromatographic method development time and reduce analysis time. Results. A full validation of the automated method is presented for a range of compounds with log D values between −2 and 4. Conclusions. The advantages and limitations of this direct measurement method are discussed. The use of this methodology provides a means to rapidly assess log D values for large compound arrays.

Parallel synthesis of 3-aryloxy-2-propanolamines and evaluation as dual affinity 5-HT1A and 5-HT re-uptake ligands

A solution phase synthesis for the preparation of 3-aryloxy-2-propanolamine libraries has been developed. This resulted in the identification of 5 as a ligand with dual affinity for 5-HT1A and serotonin reuptake receptors which shows excellent pharmacokinetic parameters.

Determination of the in vitro metabolism of (+)- and (−)-epibatidine

The oxidative in vitro metabolism of epibatidine was investigated using liver microsomes from rat, dog, rhesus monkey and human. Analysis was performed using liquid chromatography-mass spectrometry (LC-MS) using both achiral and chiral stationary phases. Comparison of the metabolism of the (+)- and (-)-enantiomers revealed species differences in the extent of metabolism, with rhesus monkey>dog>rat=human. Furthermore, differences in the routes of metabolism for epibatidine enantiomers were also observed, with mass spectra consistent with hydroxylation of the azabicycle for (-)-epibatidine and with the formation of diastereomeric N-oxides for (+)-epibatidine being obtained. For chiral LC-MS, a volatile ion-pair reagent of heptafluorobutyric acid was used in place of pentanesulphonic acid with no deterioration in chiral selectivity. Analysis of the same samples by chiral LC-MS revealed no evidence for metabolic chiral interconversion and chiral analysis from a metabolic time course of racemic material revealed enantiomers to be metabolised to approximately the same extent. Such findings may be important particularly should epibatidine be investigated in non-rodent species.

Use of chiral liquid chromatography-tandem mass spectrometry to investigate the metabolism of racemic cholecystokinin-B antagonists

In an attempt to establish the enantiomeric specificity of metabolism for a series of racemic cholecystokinin-B receptor antagonists, chiral LC-MS-MS conditions were established using a Pirkle DNBL chiral stationary phase operating in the reversed-phase mode. Rat liver microsomal incubations of the compounds were analysed using these conditions and it was demonstrated that resolution of oxygenated and demethylated metabolites could be achieved. A single model compound was investigated in detail by obtaining product-ion spectra on all mono-oxygenated species in an attempt to correlate these and identify enantiomeric pairs of metabolites. In this example a lack of differentiation in the product ion spectra did not allow correlation but the results suggest that such an approach may still be viable for the chiral metabolic analysis of racemic material.

Enantiomeric separation of substituted 2-arylindoles on derivatised polysaccharide chiral stationary phases

The enantiomeric separation of a series of 2-arylindoles, developed as 5HT(2A) receptor antagonists for the treatment of schizophrenia, was investigated. Evaluation of a number of chiral stationary phases (CSPs) suggested that Chiralcel OD-H and Chiralpak AD were the most versatile for these compounds, and were employed for more detailed studies. A degree of complementarity between the CSPs was observed, such that Chiralcel OD-H was more effective for piperidine-containing molecules and Chiralpak AD for piperazine- and morpholine-containing molecules. The presence of a basic secondary amine was detrimental chromatographically, but resolution was improved substantially by employing diethylamine (DEA) in the mobile phase. All separations were either enthalpy-controlled or showed no temperature dependence. Differential temperature effects between series highlighted the possibility of multiple binding modes on these CSPs. Based on this study, it is possible to make a more rational selection of chromatographic conditions for future novel analogues.

Chiral separation of 4,4-disubstituted piperidinyl substance P antagonists

The chiral separation of fourteen Substance P antagonists, substituted at a benzylic carbon to generate a chiral centre, was investigated using Chiracel OD-H and Chiralpak AD stationary phases. The nature of an N-substituent, distant from the chiral centre, was found to modulate separation selectivity. Aromatic substitution on the benzyl group also affected separation selectivity, but to a lesser degree. Some complementary character between Chiracel OD-H and Chiralpak AD was seen, but selection of the optimal phase did not appear predictable. The effect of temperature was also unanticipated, with some compounds showing the expected decrease of separation selectivity with temperature, whereas, an example of an improvement in separation selectivity with temperature to give an entropically controlled separation was also observed. This serves to highlight the complex nature of enantioselective interactions using chiral polymers and suggests that, given an unknown compound in this series, adequate separation conditions are difficult to predict.