This chapter contains sections titled: Introduction Development of the HTMS Method for Underivatized Cystathionine in Biological Samples Spanning In Vivo Cell Culture, and Ex Vivo Assays Development of a 2D HTMS Method for Plasma-bound Small Molecules Conclusion References

Two facile methods for preparing 4-fluororesorcinol have been developed. In the first method, the direct fluorination of 1,3-dimethoxybenzene with trifluoromethyl hypofluorite carried out in Freon 11 at −78 °C afforded 2,4-dimethoxyfluorobenzene. Demethylation by heating under reflux in 48% HBr in acetic acid gave 4-fluororesorcinol in an overall yield of 60%. The second method involved the fluorination of 2,6-dimethoxyacetophenone by trifluoromethyl hypofluorite in Freon 11 at −78 °C, leading to 2,6-dimethoxy-3-fluoroacetophenone. On heating under reflux in 48% HBr in acetic acid, 4-fluororesorcinol was obtained in an overall yield of 74%. The latter is the method of choice for preparing 4-fluororesorcinol.

Label-free mass spectrometric (MS) technologies are particularly useful for enzyme assay design for drug discovery screens. MS permits the selective detection of enzyme substrates or products in a wide range of biological matrices without need for derivatization, labeling, or capture technologies. As part of a cardiovascular drug discovery effort aimed at finding modulators of cystathionine beta-synthase (CBS), we used the RapidFire((R)) label-free high-throughput MS (HTMS) technology to develop a high-throughput screening (HTS) assay for CBS activity. The in vitro assay used HTMS to quantify the unlabeled product of the CBS reaction, cystathionine. Cystathionine HTMS analyses were carried out with a throughput of 7 s per sample and quantitation over a linear range of 80-10,000 nM. A compound library of 25,559 samples (or 80 384-well plates) was screened as singlets using the HTMS assay in a period of 8 days. With a hit rate of 0.32%, the actives showed a 90% confirmation rate. The in vitro assay was applied to secondary screens in more complex matrices with no additional analytical development. Our results show that the HTMS method was useful for screening samples containing serum, for cell-based assays, and for liver explants. The novel extension of the in vitro analytical method, without modification, to secondary assays resulted in a significant and advantageous economy of development time for the drug discovery project.

Azaindole based structures were evaluated as DP1 receptor antagonists. This work has lead to the discovery of potent, selective and distinct DP1 receptor antagonists.

A general synthesis of quinolinones and benzothiazine 1,1-dioxides is presented. A series of N-phenylacrylamides and N-phenylethylenesulfonamides were studied for their ability to undergo ring closing methathesis to yield the corresponding quinolinones and benzothiazine 1,1-dioxides, respectively. The reactions are general in scope and represents a mild method for the preparation of these heterocycles.

A new series of indole-based antagonists of the PGD(2) receptor subtype 1 (DP1 receptor) was identified and the progress of the structure-activity relationship study to the identification of potent and selective antagonists is presented. Selective DP1 antagonists with high potency and selectivity were prepared. Of particular interest is the DP1 antagonist 26 with a K(i) value of 1 nM for the DP1 receptor and an IC(50) value of 4.6 nM in a DP1 functional assay for the inhibition of the PGD(2) induced cAMP production in platelet rich plasma (PRP).

An elution—extrusion countercurrent chromatography workstation was designed and built in-house for fractionating crude natural product extracts. The engineering efforts of this project included both custom hardware and software integration. The resulting workstation operates four individual chromatography coils for natural product extract separation and purification. The workstation offers the ability to collect fractions into 16 × 100 mm borosilicate glass test tubes or allows for stream splitting of the coil effluent so that the fractions can be collected into both 16 × 100 mm borosilicate test tubes and a 96-well microplate. Solvent pumping for each of the chromatography columns is achieved through the use of syringe pumps. The workstation is controlled though a custom control software application, C_Cubed, written in Visual Basic 6.0 (VB6). Software architecture consists of three levels. At the lowest level, there are ActiveX dll device drivers that interface with the physical hardware. The middle layer is a custom scheduler allowing for multitasking of instrument movements. The upper level is composed of a Wizard-style user interface that mimics the scientific workflow. Finally, by using a software timer the software application was written to operate on a single thread, but exhibit multithreaded behavior.

A series of quinoline/naphthalene-difluoromethylphosphonates were prepared and were found to be potent PTP1B inhibitors. Most of these compounds bearing polar functionalities or large lipophilic residues did not show appreciable oral bioavailability in rodents while small and less polar analogs displayed moderate to good oral bioavailability. The title compound was found to have the best overall potency and pharmacokinetic profile and was found to be efficacious in animal models of diabetes and cancer.

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.

PTP-1B represents an attractive target for the treatment of type 2 diabetes and obesity. Given the role that protein phosphatases play in the regulation of many biologically relevant processes, inhibitors against PTP-1B must be not only potent, but also selective. It has been extremely difficult to synthesize inhibitors that are selective over the highly homologous TCPTP. We have successfully exploited the conservative Leu(119) to Val substitution between the two enzymes to synthesize a PTP-1B inhibitor that is an order of magnitude more selective over TCPTP. Structural analyses of PTP1B/inhibitor complexes show a conformation-assisted inhibition mechanism as the basis for selectivity. Such an inhibitory mechanism may be applicable to other homologous enzymes.

The Trans Cell Layer Electrical Field Stimulation (TCL-EFS) system has been developed for high-throughput screening (HTS) of voltage-gated ion channels in microplate format on a Voltage-Ion Probe Reader (VIPR) platform. In this design, a wire electrode is placed above the cell layer of each filter well, and a whole plate perimeter electrode resides beneath the filter layer. This configuration allows the electrodes to be placed away from the cell layer to minimize the near electrode field effects on cell function and dye bleaching observed with other existing designs. Mathematical simulation indicates that the electric field at the cell layer becomes uniform as the top electrode is raised to a position near the surface of the solution in the well. Using the TCL-EFS system and membrane potential fluorescence resonance energy transfer (FRET) dyes, the sensitivity of voltage-gated sodium channels to tetrodotoxin and other channel inhibitors was found to be similar to those determined by established electrophysiological and more conventional VIPR techniques. A good correlation was also observed with the TCL-EFS system for inhibition of Cav2.2 by omega-conotoxin-GVIA and for block of Cav1.2 by known small molecule inhibitors. Thus, the TCLEFS system is suitable for both quantitative analysis and HTS of voltage-gated sodium and calcium channels, without the liabilities of previously reported EFS methodologies.