Solid-phase microextraction for assessment of plasma protein binding, a complement to rapid equilibrium dialysis.

Aim: Determination of plasma protein binding (PPB) is considered vital for better understanding of pharmacokinetic and pharmacodynamic activities of drugs due to the role of free concentration in pharmacological response. Methodology & results: Solid-phase microextraction (SPME) was investigated for measurement of PPB from biological matrices and compared with a gold standard approach (rapid equilibrium dialysis [RED]). Discussion & conclusion: SPME-derived values of PPB correlated well with literature values, and those determined by RED. Respectively, average protein binding across three concentrations by RED and SPME was 33.1 and 31.7% for metoprolol, 89.0 and 86.6% for propranolol and 99.2 and 99.0% for diclofenac. This study generates some evidence for SPME as an alternative platform for the determination of PPB.

Synthesis and Spectral Characterization of Benzo-[6,7][1,5]diazocino[2,1-a]isoindol-12-(14H)-one Derivatives.

A simple synthetic route affording 27%-85% yields of benzo[6,7][1,5]diazocino[2,1-a]isoindol-12(14H)-one ring systems from readily available 3-(2-oxo-2-phenylethyl) isobenzofuran-1(3H)-ones and 2-(aminomethyl)aniline starting materials in toluene and catalysed by p-toluene-sulfonic acid is developed. The ¹H- and (13)C-NMR spectra of the final products were assigned using a variety of one and two-dimensional NMR experiments. The distinction between the two potential isomers of the final products was made on the basis of heteronuclear multiple bond connectivity (HMBC) NMR spectra.

Small molecule recognition of mephedrone using an anthracene molecular clip.

An anthracene molecular probe has been synthesised and shown to target mephedrone, a stimulant drug from the cathinone class of new psychoactive substances (NPS). A protocol has been developed to detect mephedrone via the probe using NMR spectroscopy in a simulated street sample containing two of the most common cutting agents, benzocaine and caffeine.

Direct ionization of solid-phase microextraction fibers for quantitative drug bioanalysis: from peripheral circulation to mass spectrometry detection.

A novel approach is described for the quantitative bioanalysis of drugs in blood samples by ionization of the analytes collected on solid-phase microextraction (SPME) fibers by mass spectrometry (MS). The technique combines the attractive features of SPME microsampling using minimal sample volumes with the speed, selectivity, and sensitivity capabilities of MS detection. The method reported in this study involved generating gas-phase ions directly from SPME fibers without the need for any additional sample preparation or chromatographic separation; the entire process was completed within 5 min. Traditionally, analytes extracted by SPME fibers are desorbed by washing with suitable solvents followed by a transfer into a sample vial and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to quantify the amount of analyte extracted and thereby determining the analyte concentration in the matrix. These sample preparation steps are completely eliminated by inserting the SPME fiber directly into the MS. Physiologically relevant concentrations of metoprolol and propranolol in blood samples were measured over several orders of magnitude down to concentration levels of 10 ng/mL. This preliminary assessment of direct SPME-MS showed high sensitivity (ng/mL), acceptable reproducibility (<30%), and lack of carryover. This novel approach simplifies current bioanalytical procedures providing time and cost savings. It demonstrates considerable potential for qualitative and quantitative pharmaceutical bioanalysis as well as other areas of challenging environmental and food analysis.

NK1 antagonists based on seven membered lactam scaffolds.

A new class of high affinity hNK1R antagonists based on seven-membered ring cores has been identified. This series, with relatively simple, compact structures, includes compounds with high affinity, good selectivity, and promising in vivo properties.

A new pyridazine series of GABAA alpha5 ligands.

Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.

1H NMR study of the conformation and absolute stereochemistry of two spirocyclic NK-1 antagonists.

The binding affinities at the human NK-1 receptor of two spirocyclic compounds were found to be similar despite being epimeric at a key stereocentre. This unexpected result prompted a thorough investigation of the solution conformations of the two compounds. This revealed that a conformational switch in the tetrahydrofuran ring enabled the C-3-aryl group to be equatorial in both cases, leading to a similar juxtaposition of the aryl rings.

Aryl sulfones: a new class of gamma-secretase inhibitors.

The development of a novel series of 4-aryl, 4-phenylsulfonyl cyclohexananone-derived gamma-secretase inhibitors for the potential treatment of Alzheimer's disease is described.

Open-access high-resolution mass spectrometry in early drug discovery.

Techniques such as mass spectrometry and NMR spectroscopy form an essential part of the medicinal chemist's toolbox for characterizing and assessing the purity of new molecules. Empowering medicinal chemists to gain early insight into their reaction products has a direct impact on productivity. Devolution of cutting-edge techniques from the specialist to the bench chemist also frees the specialist to concentrate on solving the more demanding of analytical problems. For open-access techniques to be taken up, they must be robust and be able to handle differing sample concentrations and varying sample complexities. This paper details the implementation of high-resolution liquid chromatography/mass spectrometry in open access to aid the medicinal chemist in characterizing desired products and identifying unexpected rearrangements, by-products and complete unknowns.

Accelerated metabolite identification by "extraction-NMR".

Examples of the use of extraction-NMR, an efficient and rapid method to obtain structural information on metabolites without prior separation, are described. Crude ethyl acetate extracts of in vitro microsomal incubations were analysed by NMR spectroscopy. The region downfield of 5.5 ppm in the proton spectra of these crude extracts was found to be essentially clear of endogenous resonances. As a consequence, sites of aromatic hydroxylation can often be determined without prior separation of the crude extracts. Sites of metabolism close to the aromatic region may also be accessible via two-dimensional (2D) homonuclear experiments (e.g. COSY, NOESY, TOCSY). One-dimensional (1D) and 2D fluorine experiments also can provide additional information on the structure of metabolites. Depending on the complexity of the aromatic region of the parent compound, signal overlap and the relative abundance of the individual components, extraction-NMR has the potential to provide information for unambiguous structure elucidation of two or three major metabolites. Should extraction-NMR produce inconclusive results, i.e. too many metabolites are present or metabolism occurred exclusively on aliphatic regions, it is possible to re-use the extraction-NMR sample and proceed with traditional methods of analysis.

Metabolite identification in drug discovery.

Recent developments in the technologies and approaches to identify metabolites in a drug discovery environment are reviewed. Samples may be generated using either in vitro systems--typically, but not exclusively, liver subcellular fractions, such as microsomes, or whole cells, such as hepatocytes. Alternatively, metabolites are generated in vivo using excreta obtained following dosing in preclinical species. Recombinant drug metabolizing enzymes or microorganisms may offer alternate vectors. New techniques, such as the use of solid-phase microextraction, have found application in the isolation of metabolites from biological matrices. However, this is still dominated by the use of preparative chromatography, which has advanced through the use of mass-directed detection. Detection and structural elucidation by mass spectrometry have improved markedly with increases in sensitivity, allowing lower abundance metabolites to be detected, and increases in selectivity, with the use of high-resolution time-of-flight and quadrupole-time-of-flight instruments. Finally, higher field strength magnets coupled with novel probe designs and increased use of liquid chromatographic hyphenation techniques continue to drive the capabilities of nuclear magnetic resonance spectroscopy as the definitive structural elucidation tool.

Synthesis and conformational dynamics of tricyclic pyridones containing a fused seven-membered ring.

A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described. These compounds exhibit atropisomerism and exist in enantiomeric forms. Chiral HPLC separation of the enantiomers has allowed the rates of racemization to be measured and hence the free energy barrier for flipping the seven-membered ring to be deduced. Introduction of a further element of planar chirality leads to diastereomeric atropisomerism. The rate of interconversion of the diastereomers has been quantified by 2D EXSY NMR spectroscopy allowing a full description of the conformational dynamics of the system.

Identification of metabolites of a substance P (neurokinin 1 receptor) antagonist in rat hepatocytes and rat plasma.

[3R,5R,6S]-3-(2-cyclopropyloxy-5-trifluoromethoxyphenyl)-6-phenyl-1-oxa-7-azaspiro[4.5]decane is a substance P (Neurokinin 1 receptor) antagonist. Substance P antagonists are proven in concept to have excellent potential for the treatment of major depression, and they allow superior and sustained protection from acute and delayed chemotherapy-induced emesis. The metabolism of this compound was investigated in rat hepatocytes, and circulating rat plasma metabolites were identified following oral and intravenous dosing. The turnover in rat hepatocytes within 4 h was about 30%, and the major metabolites were identified as two nitrones and a lactam associated with the piperidine ring. Although these metabolites were also observed in rat plasma, the major circulating metabolite was a keto acid following oxidative de-amination of the piperidine ring. Liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance were used to confirm the structure of the latter metabolite. A mechanism leading to the formation of the keto acid metabolite has been suggested, and most intermediates were observed in rat plasma.