Synthesis and SAR studies of benzimidazolone derivatives as histamine H3-receptor antagonists.

A novel series of benzimidazolone-containing histamine H3-receptor antagonists were prepared and their structure-activity relationship was explored. These benzimidazolone analogs demonstrate potent H3-receptor binding affinities, no P450 enzyme inhibition, and strong H3 functional activity. Compound 1o exhibits the best overall profile with H3Ki=0.95nM and rat AUC=12.9µMh.

Fused bicycles as arylketone bioisosteres leading to potent, orally active thiadiazole H3 antagonists.

A structure-activity relationship study was undertaken to address the lack of oral exposure of the H3 antagonist 1, which incorporated an arylketone. Among a number of sub-series, the 4H-pyrido[1,2-a]pyrimidin-4-one analog 21 showed an improved PK profile in rat and mouse and was active in an obesity model. The pyrimidin-4-one proved to be a novel and useful ketone bioisostere.

Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS.

It can be argued that the last true paradigm shift in the bioanalytical (BA) arena was the shift from high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection to HPLC with tandem mass spectrometry (MS/MS) detection after the commercialization of the triple quadrupole mass spectrometer in the 1990s. HPLC-MS/MS analysis based on selected reaction monitoring (SRM) has become the gold standard for BA assays and is used by all the major pharmaceutical companies for the quantitative analysis of new drug entities (NCEs) as part of the new drug discovery and development process. While LC-MS/MS continues to be the best tool for drug discovery bioanalysis, a new paradigm involving high-resolution mass spectrometry (HRMS) and ultrahigh-pressure liquid chromatography (uHPLC) is starting to make inroads into the pharmaceutical industry. The ability to collect full scan spectra, with excellent mass accuracy, mass resolution, 10-250 ms scan speeds and no NCE-related MS parameter optimization, makes the uHPLC-HRMS techniques suitable for quantitative analysis of NCEs while preserving maximum qualitative information about other drug-related and endogenous components such as metabolites, degradants, biomarkers and formulation materials. In this perspective article, we provide some insight into the evolution of the hybrid quadrupole-time-of-flight (Qq-TOF) mass spectrometer and propose some of the desirable specifications that such HRMS systems should have to be integrated into the drug discovery bioanalytical workflow for performing integrated qualitative and quantitative bioanalysis of drugs and related components.

Mapping pharmaceuticals in rat brain sections using MALDI imaging mass spectrometry.

Matrix-assisted laser desorption/ionization-tandem mass spectrometric method (MALDI-MS/MS) has proven to be a reliable tool for direct measurement of the disposition of small molecules in animal tissue sections. As example, MALDI-MS/MS imaging system was employed for visualizing the spatial distribution of astemizole and its primary metabolite in rat brain tissues. Astemizole is a second-generation antihistamine, a block peripheral H1 receptor, which was introduced to provide comparable therapeutic benefit but was withdrawn in most countries due to toxicity risks. Astemizole was observed to be heterogeneously distributed to most parts of brain tissue slices including cortex, hippocampus, hypothalamic, thalamus, and ventricle regions while its major metabolite, desmethylastemizole, was only found around ventricle sites. We have shown that astemizole alone is likely to be responsible for the central nervous system (CNS) side effects when its exposures became elevated.

The synthesis and structure-activity relationship of 4-benzimidazolyl-piperidinylcarbonyl-piperidine analogs as histamine H3 antagonists.

A structure-activity relationship study of the lead piperazinylcarbonylpiperidine compound 3 resulted in the identification of 4-benzimidazolyl-piperidinylcarbonyl-piperidine 6h as a histamine-3 (H(3)) receptor antagonist. Additional optimization of 6h led to the identification of compounds 11i-k with K(i)

The role of exploratory drug metabolism and pharmacokinetics in new drug research: case study-selection of a thrombin receptor antagonist for development.

The rising costs and time associated with bringing new medicines to the market have created a need for a new paradigm for reducing the attrition rates of drug candidates in both preclinical and clinical development stages. Early appraisal of drug metabolism and pharmacokinetic (DMPK) parameters is now possible due to several higher throughput in vitro and in vivo screens. This knowledge of DMPK properties should not only shorten the timelines for the selection of drug candidates but also enhance the probability of their success for development. The role of DMPK researchers in the drug research paradigm should not be limited to screening a large array of compounds during the lead optimization process but should include a strive for an understanding of the absorption, distribution, metabolism, excretion, and potential drug-related toxicities of a chemical series. As an example, in this article we present a specific DMPK research screening paradigm and describe a case study using the Thrombin Receptor Antagonist program. This screening paradigm followed by the extensive lead optimization process culminated in the selection of SCH 530348, a potent, selective and orally active thrombin receptor antagonist for the treatment of thrombosis.

Development of a biomarker assay for 3-indoxyl sulfate in mouse plasma and brain by liquid chromatography/tandem mass spectrometry.

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric method for the determination of 3-indoxyl sulfate (3-IS), an endogenous compound in mammals, in mouse plasma and brain samples. The analytical method involves direct dilution of samples with water and protein precipitation with acetonitrile containing an internal standard, followed by separation of 3-IS on a MonoChrom C(18) column and detected by selected reaction monitoring (SRM) in negative ionization mode using turbo ion-spray ionization. Due to high endogenous levels of 3-IS in control mouse plasma and brain, blank guinea pig plasma and brain were used for the preparation of standard curves and quality controls (QCs). The compound of interest was well separated from interference peaks from the matrices with a total runtime of 2.7 min under a gradient condition. The method was partially validated. The linear concentration range was 0.1 to 100 microg/mL in mouse plasma and 10 to 10,000 ng/g in mouse brain. Inter-assay mean bias and relative standard deviation (RSD) for plasma were in the range of -4.8% to 3.1% and 2.5% to 3.2%, respectively. Intra-assay mean bias and RSD for plasma were in the range of -3.3% to 1.4% and 1.9% to 2.8%, respectively. Inter-assay mean bias and RSD for brain were in the range of -1.8% to 3.5% and 1.7% to 8.1%, respectively. Intra-assay mean bias and RSD for brain were in the range of -1.7% to 3.9% and 4.1% to 7.3%, respectively. The lower limit of quantitation (LLOQ) for this assay was 0.1 microg/mL for plasma and 10 ng/g for brain. The matrix effect was not observed in both guinea pig plasma and mouse plasma.

Advances in the integration of drug metabolism into the lead optimization paradigm.

The lead optimization paradigm includes a team of experts that has a multitude of parameters to consider when moving from an initial lead compound through the lead optimization phase to the development phase. While in the past the team may have had only a medicinal chemist and a pharmacologist, the current team would often include experts in the areas of drug metabolism and pharmacokinetics (DMPK) as well as chemical toxicity. This review provides an overview of the some of the recent advances in the areas of DMPK screening plus a discussion of some of the assays that can be used to begin to screen for toxicity issues. The focus of this review is the major potential problem areas: oral bioavailability, half-life, drug-drug interactions and metabolism and toxicity issues.

MALDI-tandem mass spectrometry imaging of astemizole and its primary metabolite in rat brain sections.

BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI)-tandem mass spectrometry (MS)/MS is a proven reliable tool for visualizing the spatial distribution of dosed drugs and their primary metabolites in animal tissue sections. MATERIALS & METHODS: The rat brain tissue sections coated with dihydroxybenzoic acid as matrix, were analyzed by MALDI-MS/MS imaging experiments. The potential metabolites of astemizole in rat brain homogenate selected for MALDI-MS/MS imaging experiments were first identified by high-performance liquid chromatography coupled to an electrospray ionization source and a hybrid-quadrupole-linear-ion-trap mass spectrometer. RESULTS: Astemizole was observed to be heterogeneously distributed to most parts of the brain tissue slices including the cortex, hippocampus, hypothalamic, thalamus and ventricle regions, while its major metabolite, desmethylastemizole, was only found around ventricle sites. CONCLUSION: The results indicated that the dosed compound alone might be responsible for the CNS side-effects when drug exposures became elevated.

High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometry for the detection of 17alpha-ethinylestradiol in hepatocytes.

Atmospheric pressure photoionization (APPI) as an interface for the high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) system was employed for the direct determination of 17alpha-ethinylestradiol (EE(2)) in the incubation mixtures to support in vitro hepatic clearance studies. For the APPI source, the radical cation of the analyte via charge exchange with the dopant radical cation was used for the detection of EE(2) in the positive ion mode. It was demonstrated that the major signals of EE(2) in the acetonitrile/water mobile phase were substantially increased by replacing toluene with anisole as the dopant. The effects of several experimental conditions on the photoionization efficiency of EE(2) in the dopant-assisted APPI source were explored. Electrospray ionization (ESI) source was also suitable for the analysis of the analyte; however, ESI required a derivatization step prior to analysis. The applicability of the proposed HPLC-APPI-MS/MS approach following a protein precipitation procedure for the determination of EE(2) at low nano-mole levels was examined with respect to assay specificity and linearity. The assay results obtained by both HPLC-APPI-MS/MS and HPLC-ESI-MS/MS methods were in good agreement.

Visualization of first-pass drug metabolism of terfenadine by MALDI-imaging mass spectrometry.

The aim of this article is to focus on the implementation and the application of matrix-assisted laser desorption/ionization-imaging mass spectrometric system (MALDI-IMS) to determine the disposition or biotransformation pathway of terfenadine and its active metabolite, fexofenadine in mouse and rat whole-body tissue sections. Whole-body MALDI-IMS data showed that the poor oral bioavailability of terfenadine was largely due to high first-pass metabolism in the intestines and the liver before the compound reached systemic circulation.

Ultra-performance liquid chromatography/tandem mass spectrometric determination of diastereomers of SCH 503034 in monkey plasma.

This paper describes the development and qualification of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of diastereomers of SCH 503034 in monkey plasma. The analytical method involves direct protein precipitation with a mixture of methanol/acetonitrile (10/90) containing an internal standard, followed by separation of the stereoisomers on an Acquity UPLC C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using atmospheric pressure chemical ionization (APCI). The effects of ion-pairing agents on separation and ionization efficiency were investigated. The two diastereomers were well separated (R=1.3) with a runtime of 5 min under an isocratic condition. The method was qualified. The linear concentration range was 1-2500 ng/ml for the both stereoisomers. Inter-assay mean bias and relative standard deviation (R.S.D.) were in the range of -1.2% to 3.6% and 2.8-10%, respectively. Intra-assay mean bias and R.S.D. were in the range of -1.3% to 5.5% and 2.3-7.8%, respectively. Recoveries of the stereoisomers at concentration levels of 2.5, 50 and 1000 ng/ml were 87.2-90.0%, 89.1-90.4% and 92.3-94.3%, respectively. The LLOQ for this assay was 1 ng/ml. No matrix interferences were observed in six different sources of blank monkey plasma.

Lead Optimization in Discovery Drug Metabolism and Pharmacokinetics/Case study: The Hepatitis C Virus (HCV) Protease Inhibitor SCH 503034.

Lead optimization using drug metabolism and pharmacokinetics (DMPK) parameters has become one of the primary focuses of research organizations involved in drug discovery in the last decade. Using a combination of rapid in vivo and in vitro DMPK screening procedures on a large array of compounds during the lead optimization process has resulted in development of compounds that have acceptable DMPK properties. In this review, we present a general screening paradigm that is currently being used as part of drug discovery at Schering-Plough and we describe a case study using the Hepatitis C Virus (HCV) protease inhibitor program as an example. By using the DMPK optimization tools, a potent HCV protease inhibitor, SCH 503034, was selected for development as a candidate drug.

Mapping pharmaceuticals in tissues using MALDI imaging mass spectrometry.

During drug discovery and development stage, often the question is raised as to whether the drug can reach the site of action which helps researchers better assess the potential value of that compound as a pharmaceutical product and toxicological outcomes. High performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS) has totally replaced HPLC methods that use UV or other detectors for most drug analysis applications. However, HPLC-MS/MS approaches are not able to provide the answer to certain questions regarding the distribution of a drug in various organs or tissues from laboratory animal experiments. Whole body radioautography (WBA) normally provides a standard means to answer this question on the time course of the drug candidates. However, the major disadvantage in this radioautographic technique is to allow for visualization of total drug-related materials but to image the distribution of the administrated drugs and their metabolites in all tissues. In addition, the availability of radiolabeled compounds at drug discovery stage is another concern. To overcome these issues, matrix-assisted laser desorption/ionization-mass spectrometric method (MALDI-MS) has been developed to directly determine the distribution of pharmaceuticals in tissue sections which might unravel their disposition or biotransformation pathway for new drug development.

Ultra-performance liquid chromatography/tandem mass spectrometric determination of testosterone and its metabolites in in vitro samples.

This paper describes the development and partial validation of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of testosterone (T) and its four metabolites, 6beta-OH-T, 16alpha-OH-T, 16beta-OH-T and 2alpha-OH-T, in in vitro samples. The analytical method involves direct dilution of samples with acetonitrile containing an internal standard, followed by separation of testosterone and the four metabolites on an Acquity UPLCtrade mark C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using turbo ionspray ionization. The parent compound and its metabolites investigated were well separated (Rs >1.5) with a run time of 4 min under a gradient condition. The method was partially validated. The linear concentration range was 0.01 to 5 microM for all the compounds of interest. Inter-assay mean bias and relative standard deviation (RSD) were in the range of -12% to 8% and 4.1% to 8.5%, respectively. Intra-assay mean bias and RSD were in the range of -8.0% to 5.2% and 3.4% to 9.6%, respectively. The lower limit of quantitation for this assay was 0.01 microM. The differences in LC/MS performance were investigated by conducting a comparison of UPLC with another method previously optimized for HPLC-based separation and quantification of testosterone and its metabolites.

Increasing speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic screening.

Both combinatorial chemistry and parallel synthesis provide a valuable means for the production of large numbers of compounds with diverse molecular architectures that become available for various drug discovery experiments. In both the lead optimization and lead selection stages, one requirement that is common for many processes is the need for bioanalytical support. This review summarizes current high throughput strategies and efficient methodologies that are employed for drug metabolism and pharmacokinetic (DMPK) screens for a series of drug discovery compounds. For these types of assays, high performance liquid chromatography coupled to a tandem mass spectrometer (HPLC-MS/MS) has now become the technique of choice. The major high throughput strategies including sample reduction and cassette dosing are discussed. The methods for increasing the speed of HPLC-MS/MS-based analyses, such as fast chromatography, direct sample injection, parallel technologies and combined ionization interfaces are also presented in this review. In addition, the special challenges when performing HPLC-MS/MS bioanalysis, such as the choice of ionization sources, matrix ionization suppression and the potential for endogenous interferences, are addressed.

Supercritical fluid chromatography-tandem mass spectrometry for the enantioselective determination of propranolol and pindolol in mouse blood by serial sampling.

Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimum sample pretreatment was explored for the rapid and enantioselective determination of (R,S)-propranolol in mouse blood. Serial bleeding of mice is advantageous for the reduction of animal usage, dosing errors, and animal-to-animal variation. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the ionization efficiency of racemic propranolol and pindolol as model compounds in the positive ion mode under pSFC conditions were studied. The fundamental parameters on the proposed hyphenated system such as matrix ionization suppression and chromatographic performances were investigated in improving sensitivity and enantiomeric separation for the detection of the analytes. The proposed chiral pSFC-APCI/MS/MS approach requiring approximately 3 min/sample for the determination of (R,S)-propranolol at a low nanogram per milliliter region was partially validated with respect to specificity, linearity, reproducibility, and accuracy and was applied to support a pharmacokinetic study.

Fast mass spectrometry-based methodologies for pharmaceutical analyses.

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.

Investigation of the profiling depth in matrix-assisted laser desorption/ionization imaging mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry is generally considered to be a surface analysis technique. In this report, the profiling depth of imaging mass spectrometry was examined. MALDI matrix solution was found to be able to gain access to the tissue interior and extract analyte molecules to the tissue surface. As a consequence, prazosin, a small molecule pharmaceutical compound, located as deep as 40 microm away from the surface was readily detected after matrix application. Likewise, cytochrome c, a 12 kDa protein, was also detectable from the tissue interior. Moreover, for prazosin, not only the extent of matrix effect, but also the extraction efficiency of the matrix solvent appeared to be dependent on the type of tissue. These results indicated that experimental conditions that decrease the matrix solvent evaporation during matrix application may increase analyte extraction efficiency and hence sensitivity of the analysis. Furthermore, thin sections should be used to avoid differential extraction efficiency of matrix solvent in different tissues for whole-body analysis.