2-Aryl indole NK1 receptor antagonists: optimisation of indole substitution.

The synthesis and biological evaluation of a series of 2-aryl indoles with high affinity for the human neurokinin-1 (hNK1) receptor are reported, concentrating on optimisation of the indole substitution.

Quantitative determination of L-775,606, a selective 5-hydroxytryptamine 1D agonist, in rat plasma using automated sample preparation and detection by liquid chromatography-tandem mass spectrometry.

L-775,606 is under investigation as a selective 5-hydroxytryptamine 1D agonist for the treatment of migraine. A reliable and sensitive method for the analysis of L-775,606 in plasma was required in order to support preclinical evaluation of this compound. A semi-automated sample preparation method using the Beckman Biomek 2000 workstation to perform all liquid handling tasks has been established. The sample analysis was performed using HPLC-MS-MS with a cycle time of 3.5 min per sample. Intra- and inter-day assay accuracy and precision are excellent with a calibration range of 1-2000 ng/ml and a reproducible limit of quantification of 1 ng/ml.

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.

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.

Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS.

Generic methodology for the automated preparation and analysis of drug levels in plasma samples within a drug discovery environment was achieved through the redesign of a protein precipitation assay to a microtiter (96-well) plate format and the application of robotic liquid handling for performance of all transfer and pipetting steps. Validation studies revealed that the application of robotics to sample preparation, in general, maintained the analytical accuracy and precision compared with preparing samples manually. The use of rapid gradient LC-MS/MS for analysis coupled with flow diversion of the solvent front allowed the introduction of protein-precipitated samples into the mass spectrometer without the necessity for source cleaning. The problem inherent in automatically pipetting plasma, caused by fibrinogen clots, was overcome by storing samples at -80 degrees C and thus precluding clot formation. The resulting methodology allowed sample preparation for a 96-well plate designed to accommodate 54 unknowns, duplicate 12-point calibration curves, and 6 sets of quality controls at three levels in approximately 2 h. This approach allowed an increase in throughput of sample preparation and analysis to >400 samples per day per LC-MS/MS instrument with minimal manual intervention. Overall, substantial time savings were realized, demonstrating that automation is an increasingly essential tool in a drug discovery bioanalytical environment.

An increased throughput method for the determination of partition coefficients.

PURPOSE: 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 autosampler 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.

Fluorination of 3-(3-(piperidin-1-yl)propyl)indoles and 3-(3-(piperazin-1-yl)propyl)indoles gives selective human 5-HT1D receptor ligands with improved pharmacokinetic profiles.

It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.