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Antagonism of the adenosine A(2a) receptor offers great promise in the treatment of Parkinson's disease. In the course of exploring pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A(2A) antagonists, which led to clinical candidate SCH 420814, we prepared 1,2,4-triazolo[1,5-c]pyrimidines with potent and selective (vs A(1)) A(2a) antagonist activity, including oral activity in the rat haloperidol-induced catalepsy model. Structure-activity relationships and plasma levels are described for this series.

SCH 58261 is a reported adenosine A2A receptor antagonist, which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A1 receptor and does not demonstrate oral activity. We report the design and synthesis of biaryl and heteroaryl analogs of SCH 58261 which improve the A2A receptor binding selectivity as well as the pharmacokinetic properties of SCH 58261. In particular, the quinoline 25 has excellent A2A receptor in vitro binding affinity and selectivity, sustained rat plasma levels upon oral dosing, and is active orally in a rat behavioral assay.

SCH 58261 is a reported adenosine A(2A) receptor antagonist which is active in rat in vivo models of Parkinson's Disease upon ip administration. However, it has poor selectivity versus the A(1) receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A(2A) receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A(2A) receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson's Disease, and has aqueous solubility of 100 microM at physiological pH.

Antagonism of the adenosine A2A receptor offers great promise in the treatment of Parkinson's disease. Employing the known pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine A2A antagonist SCH 58261 as a starting point, we identified the potent and selective (vs. A1) antagonist 11h, orally active in the rat haloperidol-induced catalepsy model. We further optimized this lead to the methoxyethoxyethyl ether 12a (SCH 420814), which shows broad selectivity, good pharmacokinetic properties, and excellent in vivo activity.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to directly analyze and image pharmaceutical compounds in intact tissue. The anti-tumor drug SCH 226374 was unambiguously determined in mouse tumor tissue using MALDI-QqTOFMS (QSTAR) by monitoring the dissociation of the protonated drug at m/z 695.4 to its predominant fragment at m/z 228.1. A second drug, compound A, was detected in slices of rat brain tissue following oral administration with doses ranging from 1-25 mg/kg. Quantitation of compound A from whole brain homogenates using routine high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedures revealed that concentrations of the drug in whole brain varied from a low of 24 ng/g to a high of 1790 ng/g. The drug candidate was successfully detected by MALDI-QqTOF in samples from each dose, covering a range of approximately two orders of magnitude. In addition, good correlation was observed between the MALDI-QqTOFMS intensities at each dose with the HPLC/MS/MS results. Thus the MALDI-MS response is proportional to the amount of drug in tissue. Custom software was developed to facilitate the imaging of small molecules in tissue using the MALDI-QqTOF mass spectrometer. Images revealing the spatial localization of SCH 226374 in tumor tissue and compound A in brain tissue were acquired.

A high-throughput semi-automated procedure for simultaneously stability evaluation of multiple compounds in plasma using direct single column high-performance liquid chromatography (HPLC) combined with tandem mass spectrometry (MS/MS) was developed to eliminate the laborious procedures that are traditionally used for stability studies. Untreated human, monkey, mouse and rat plasma samples containing ten drug components were directly injected into a mixed-functional column that provided both protein removal and chromatographic functionality. Ten test compounds were simultaneously assayed using a tandem mass spectrometer in the positive ion mode using multiple reaction monitoring (MRM). Plasma samples containing ten test compounds were placed in a thermostatic autosampler and then sequentially monitored in one analytical procedure. The time between each injection was set about 7 minutes. The peak responses of the test compounds in individual plasma samples were repeatedly determined every 28 minutes. Drug stability in plasma was indicated by the change of the mass chromatographic peak areas for the test compounds and was observed to be a function of animal species, incubation time and incubation temperature. The potential for matrix ionization suppression on the direct single column HPLC-MS/MS system was also investigated using the post-column infusion technique. The proposed cassette assay procedure provides an analytical throughput ten times greater than the single component approach for the evaluation of drug stability in plasma without compromising data quality.

A series of studies was performed to investigate some of the causes for matrix effects ('ion suppression' or 'ion enhancement') in bioanalytical high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assays. Previous studies have reported that matrix effects are mainly due to endogenous components in biological fluids and are a greater concern for electrospray ionization (ESI) than for atmospheric pressure chemical ionization (APCI). In this report we demonstrate that: (1) matrix effects can also be caused by exogenous materials, such as polymers contained in different brands of plastic tubes, or Li-heparin, a commonly used anticoagulant; (2) matrix effects are not only ionization mode (APCI or ESI) dependent, but also source design (Sciex, Finnigan, Micromass) dependent; and (3) for at least one vendor's design, we found the APCI mode to be more sensitive to matrix effects than the ESI mode. Based on these findings, we have proposed the following simple strategies to avoid matrix effects: (1) select the same brand of plastic tubes for processing and storing plasma samples and spiked plasma standards; (2) avoid using Li-heparin as the anticoagulant; and (3) try switching the ionization mode or switching to different mass spectrometers when matrix effects are encountered. These three strategies have allowed us to use protein precipitation and generic fast LC techniques to generate reliable LC/MS/MS data for the support of pharmacokinetic studies at the early drug discovery stage.

The metabolism of SCH 40120, which is the clinically effective antipsoriatic drug 10-(3-chlorophenyl)-6,8,9,10-tetrahydrobenzol[b][1,8]naphthyrid in-5(7H)-one, was determined in vitro. Rat, dog, cynomolgus monkey, and human liver slices hydroxylated the aliphatic, cyclohexenyl ring of the drug and conjugated the resulting carbinol. The identified metabolites comprised the corresponding 6-, 7-, and 9-carbinols, the glucuronide of the 6-carbinol, and the 6-ketone derived from the parent drug. Although the three carbinols appeared in the liver isolates of all species studied, the relative amounts of these metabolites varied across species. With a high, non-physiological ratio of substrate to liver, the 6-carbinol and its glucuronide were the major metabolites in human and monkey, whereas the 6-ketone was a minor metabolite in dog. Containing a stereogenic axis and center, the 6-carbinol existed as diastereomeric atropisomers. Its structure was established by 13C and 1H NMR spectroscopy, mass spectrometry, and comparison to an authentic sample.

HPLC combined with atmospheric pressure ionization (API) mass spectrometry (MS) has become a very useful tool in the pharmaceutical industry. The technique of HPLC-API/MS/MS is becoming very important for both drug discovery and drug development programs. In the drug discovery area, it has three major uses: (1) rapid, quantitative method development, (2) metabolite identification, and (3) multi-drug analysis. The sensitivity of the API source and the selectivity provided by tandem mass spectrometry (MS/MS) enable rapid, quantitative method development for drugs in plasma. Early information on the metabolism of candidate drugs can guide structural modifications, thereby improving the activity and/or bioavailability.

A rapid bioanalytical method was evaluated for the simultaneous determination of drug discovery compounds and their potential metabolites in plasma samples within 1 min run time by fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). The fast HPLC-MS/MS system is achieved by using mini-column HPLC coupled to tandem mass spectrometer which is advantageous over regular HPLC-MS/MS systems, such as a shorter chromatographic region of ionization suppression, less solvent consumption and higher throughput. Matrix ionization suppression effect of the test compounds in plasma samples when using fast HPLC-MS/MS method was examined by a post-column infusion technique. In the described example, the proposed approach has been successfully employed to determine the plasma concentration of the test compound and its hydroxyl metabolite (M+16) in monkey in the low ng/ml region. The monkey pharmacokinetic results obtained by the proposed fast HPLC-MS/MS method were in good agreement within 20% error with those obtained by the regular HPLC-MS/MS method based on the same sample preparation procedure.

A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS–MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 °C, but it could be stabilized by adding sodium thiosulfate.

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.

A polymer-coated mixed-function (PCMF) column was evaluated for direct plasma injection for the simultaneous determination of a drug candidate and its hydroxyl metabolite by high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS-MS) in support of pharmacokinetic studies. Each diluted monkey plasma sample containing internal standard was directly injected on to the PCMF column for sample clean-up, enrichment and chromatographic separation. The proteins and macromolecules were first eluted from the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column were then eluted with a strong mobile phase using a gradient separation technique at a constant flow rate of 1.0 ml min−1. When not diverted, the column effluent was connected either to the atmospheric pressure chemical ionization (APCI) source or the electrospray ionization (ESI) source as part of the mass spectrometer system used for quantification. The calibration curve was linear over the range 5–2500 ng ml−1 for both analytes. The retention times for the analytes and the internal standard were both consistent and no column deterioration was observed for at least 500 injections. The recovery through the column and reproducibility of the dosed compound and its hydroxyl metabolite in monkey plasma samples were >90% (RSD

A higher-throughput bioanalytical method based on fast-gradient (1 min run time) high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen-type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post-column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome.

A direct injection method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for the simultaneous determination of two drug candidates in monkey plasma samples in support of pharmacodynamic studies. Each diluted monkey plasma sample containing internal standard was directly injected into a mixed-function column for sample cleanup, enrichment and chromatographic separation. The proteins and macromolecules first passed through the column while the drug molecules were retained on the bonded hydrophobic phase. The analytes retained on the column with an aqueous liquid mobile phase were then chemically eluted by switching to a strong organic mobile phase at a constant flow rate of 1.0 ml/min. The column effluent was also diverted from waste to mass spectrometer for analyte detection. Samples from two different analyte studies were assayed in one analytical procedure and the calibration curves were prepared using both analytes. The calibration curves were linear over the range of 5–2500 ng/ml for both analytes. The retention times for analytes and the internal standard were found to be consistent and no column deterioration was observed after 200 injections. The apparent on-column recoveries for the test compounds in monkey plasma samples were greater than 90% with 6% CV ( N =5). The total analysis time was less than 5 min per sample.

The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes.

A series of biologically active steroids was analyzed by both, ‘filament on’ thermospray using 100% methanol as the mobile phase with ammonium acetate as buffer, and direct chemical ionization with NH3 gas. The mass spectra obtained were very similar. Addition of pyridine instead of ammonium acetate showed selectivity in adding to the steroid molecule.