Identification of 3-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists.

As part of our efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands for this purpose. Herein we report the identification of a novel series of 3-sulfonylindazole derivatives with acyclic amino side chains as potent and selective 5-HT(6) antagonists. The synthesis and detailed SAR of this class of compounds are reported.

Advancing the FDA/Office of Regulatory Affairs Mycotoxin Program: New Analytical Method Approaches to Addressing Needs and Challenges.

The U.S. Food and Drug Administration (FDA), Office of Regulatory Affairs (ORA) oversees FDA field laboratories, monitoring the occurrence and levels of toxic mycotoxins in domestic and imported human and animal food products that have the potential to impact human and animal health when consumed. The mycotoxins being routinely monitored in human and animal foods and feeds by the Agency include aflatoxins (B1, B2, G1, G2, and M1), fumonisins (FB1, FB2, and FB3), deoxynivalenol, ochratoxin A, patulin, and zearalenone. There has been an ongoing expansion of the Sample Collection Operation Planning Effort (SCOPE) for the mycotoxin program to monitor more mycotoxins in a wider variety of food and feed matrices. To meet this pressing need, we are in the process of modernizing and harmonizing the FDA/ORA mycotoxin program in the field laboratories using approaches such as adopting new analytical technologies/methods to further advance the service. This short perspective gives an overview of the FDA mycotoxin program in the field laboratories and the current program status, discusses the need to advance the program, strategies for modernization and harmonization by implementing liquid chromatography-mass spectrometry technologies for multi-mycotoxin analysis, benefits of doing this, and challenges in taking this new approach. Perspectives on finding solutions to tackle challenges and addressing emerging issues are also discussed.

Suitability of tetrahydofuran as a dopant and the comparison to other existing dopants in dopant-assisted atmospheric pressure photoionization mass spectrometry in support of drug discovery.

In this paper, we investigated the suitability of tetrahydofuran (THF) as a dopant and compared it against other common dopants for atmospheric pressure photoionization mass spectrometry (APPI-MS). In a systematic analysis of 37 drug standards and 100 Wyeth proprietary drug candidates, THF was found to increase ionization efficiency as high as 33-fold when introduced through a syringe pump at a flow rate of 20 microL/min, and as high as 114-fold when introduced through the mobile phase at 100 microL/min. As a dopant, THF is as effective as acetone, better than anisole, and slightly less effective than toluene for the majority of the test compounds. The increase in ionization efficiency by THF was found to be compound-dependent. THF was more effective in facilitating the ionization of polar compounds than of non-polar compounds. With THF, toluene and acetone as dopants, a single type of molecular ion ([M+H](+) or M(+*)) is produced for analyte molecules. However, anisole can cause the formation of an ion cluster for polar analytes. The cluster contains [M-2H+H](+), M(+*), and [M+H](+) ions with varied ratios. This complexity may make interpretation of spectra difficult for unknown compounds when complimentary data are not available. Our findings indicate that THF is a suitable dopant in the daily usage for increasing ionization efficiency, especially when THF is used as the mobile phase or as an organic modifier in the mobile phase.

A Collaborative Study: Determination of Mycotoxins in Corn, Peanut Butter, and Wheat Flour Using Stable Isotope Dilution Assay (SIDA) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

A collaborative study was conducted to evaluate stable isotope dilution assay (SIDA) and LC-MS/MS for the simultaneous determination of aflatoxins B1, B2, G1, and G2; deoxynivalenol; fumonisins B1, B2, and B3; ochratoxin A; HT-2 toxin; T-2 toxin; and zearalenone in foods. Samples were fortified with 12 13C uniformly labeled mycotoxins (13C-IS) corresponding to the native mycotoxins and extracted with acetonitrile/water (50:50 v/v), followed by centrifugation, filtration, and LC-MS/MS analysis. In addition to certified reference materials, the six participating laboratories analyzed corn, peanut butter, and wheat flour fortified with the 12 mycotoxins at concentrations ranging from 1.0 to 1000 ng/g. Using their available LC-MS/MS platform, each laboratory developed in-house instrumental conditions for analysis. The majority of recoveries ranged from 80 to 120% with relative standard derivations (RSDs) <20%. Greater than 90% of the average recoveries of the participating laboratories were in the range of 90-110%, with repeatability RSDr (within laboratory) < 10% and reproducibility RSDR (among laboratory) < 15%. All Z scores of the results of certified reference materials were between -2 and 2. Using 13C-IS eliminated the need for matrix-matched calibration standards for quantitation, simplified sample preparation, and achieved simultaneous identification and quantitation of multiple mycotoxins in a simple LC-MS/MS procedure.

Rearrangement of 3,3-disubstituted indolenines and synthesis of 2,3-substituted indoles.

Synthesis of 2,3-substituted indoles from phenylhydrazine and alpha-branched aldehydes via rearrangement of 3,3-disubstituted indolenine intermediates is reported. [reaction: see text]

Trace LC/MS/MS quantitation of 17beta-estradiol as a biomarker for selective estrogen receptor modulator activity in the rat brain.

A sensitive LC/MS/MS method has been developed by derivatization of 17beta-estradiol (E2) with dansyl chloride to quantitate 17beta-E2 in female rat serum. The use of E2-d(5) minimized interferences from endogenous 17beta-E2 in order to achieve a limit of quantitation (LOQ) of 2.5 pg/ml using 150 microl of female rat serum. The recovery of the dansyl derivative was 95% or greater in quality control samples. The intra and interday assay precision was better than 8.2 and 6.2%, respectively, with accuracies ranging from 97 to 101% in the quality control samples. The assay was used for the quantitation of serum E2 as a biomarker for the estrogen receptor (ER) antagonist activity of small molecule SERMs (selective estrogen receptor modulators) in the female rat brain. The study revealed that a statistically significant upregulation of serum 17beta-E2 occurred for rats dosed with SERMs that are known to penetrate the brain and disrupt the hypothalamic-pituitary-ovarian (HPO) axis. Variations in 17beta-E2 in ascending dose studies also correlated with the corresponding trends in CYP17a1 levels, an mRNA biomarker for ovarian hyperstimulation. This biomarker assay has provided a useful screen for medicinal chemistry optimization to produce SERMs that do not interfere with negative feedback of estrogens on the brain and for biological hypothesis testing.

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes.

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.

Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery.

The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.