The synthesis and biological evaluation of quinolyl-piperazinyl piperidines as potent serotonin 5-HT1A antagonists.

As part of an effort to identify 5-HT(1A) antagonists that did not possess typical arylalkylamine or keto/amido-alkyl aryl piperazine scaffolds, prototype compound 10a was identified from earlier work in a combined 5-HT(1A) antagonist/SSRI program. This quinolyl-piperazinyl piperidine analogue displayed potent, selective 5-HT(1A) antagonism but suffered from poor oxidative metabolic stability, resulting in low exposure following oral administration. SAR studies, driven primarily by in vitro liver microsomal stability assessment, identified compound 10b, which displayed improved oral bioavailability and lower intrinsic clearance. Further changes to the scaffold (e.g., 10r) resulted in a loss in potency. Compound 10b displayed cognitive enhancing effects in a number of animal models of learning and memory, enhanced the antidepressant-like effects of the SSRI fluoxetine, and reversed the sexual dysfunction induced by chronic fluoxetine treatment.

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Hybrid triple quadrupole-linear ion trap mass spectrometry in fragmentation mechanism studies: application to structure elucidation of buspirone and one of its metabolites.

The use of a hybrid triple quadrupole-linear ion trap (QqQ(LIT)) mass spectrometer system for a comprehensive study of fragmentation mechanisms is described. The anxiolytic drug, buspirone, was chosen as a model compound for this study. With the advent of a QqQ(LIT) instrument, both the traditional quadrupole and the new linear ion trap scans (LIT) could be performed in a single LC run. In the past, a sample had to be run on two different instruments, namely, a triple quadrupole instrument (QqQ) and a 3D ion trap (3D IT) to obtain similar information. With the new QqQ(LIT) technology, collision-induced dissociation (CID) occur in a quadrupole collision cell, q2, and fragment ions are trapped and analyzed in Q3 operated in LIT mode. In this work, high-sensitivity product ion spectra of buspirone were obtained from the one-stage 'Enhanced Product Ion' scan (EPI) with rich product ions and no low mass cut-off. Furthermore, detailed fragmentation pathways were elucidated by further dissociation of each of the fragment ions in the EPI spectrum using MS(3) mode in the same run. The MS(3) scan was performed by incorporating CID in q2, and trapping, cooling, isolation, and resonance-excitation in Q3 when operating in LIT mode. This approach allowed unambiguous assignment of all fragment ions quickly with fewer experiments and easier interpretation than the previous approach. The overall sensitivity for obtaining complete fragment ion data was significantly improved for QqQ(LIT) as compared with that of QqQ and 3D IT mass spectrometers. This is beneficial for structure determination of unknown trace components. The method allowed structure determination of metabolites of buspirone in rat microsomes at 1 microM concentration, which was a 10-fold lower concentration than was needed for QqQ or 3D IT instruments. The QqQ(LIT) instrument provided a simple, rapid, sensitive and powerful approach for structure elucidation of trace components.

Integrated high capacity solid phase extraction-MS/MS system for pharmaceutical profiling in drug discovery.

A method is described for use in analysis of samples from pharmaceutical profiling of early drug discovery compounds. The method consists of a high capacity autosampler which injects samples into one of two solid phase extraction columns operated in parallel for alternating trapping, washing and elution into a tandem quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) MS/MS mode. A primary method, which is useful for 80-90% of compounds, and a secondary method, which is useful for a majority of the remaining compounds, are described. No analytical HPLC column is used and the analysis rate is approximately 50 samples/h. Specificity is obtained using MRM analysis. Application of the method for high capacity analysis of metabolic stability samples is described.

Optimization of a higher throughput microsomal stability screening assay for profiling drug discovery candidates.

Metabolic stability plays an important role in the success of drug candidates. First-pass metabolism is one of the major causes of poor oral bioavailability and short half-life. Traditionally, metabolic stability was evaluated at a later stage of drug discovery and required laborious manual manipulations. With the advance of high-throughput screening, combinatorial chemistry, and early profiling of drug-like properties, automated and rapid stability assays are needed to meet the increasing demand of throughput, speed, and reproducibility at earlier stages of drug discovery. The authors describe optimization of a simple, robust, high-throughput microsomal stability assay developed in a 96-well format. The assay consists of 2 automated components: robotic sample preparation for incubation and cleanup and rapid liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis to determine percent remaining of the parent compound. The reagent solutions and procedural steps were optimized for automation. Variables affecting assay results were investigated. The variability introduced by microsome preparations from different sources (various vendors and batches) was studied and indicates the need for careful control. Quality control and normalization of the stability results are critical when applying the screening data, generated at different times or research sites, to discovery projects.

Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography-mass spectrometry.

A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C(18) column is used with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus LogD(7.4) (literature). The correlation of LogD(7.4) (literature) to LogD(7.4) (HPLC) for 60 structurally diverse drugs has a correlation coefficient r(2) of 0.89. The method is applicable to compounds with MW>200 and retention time>1.5 min for rapid, initial pharmaceutical profiling in drug discovery.