Alvin C. Bach

Nanoprobe NMR spectroscopy and in vivo microdialysis: new analytical methods to study brain neurochemistry.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the chemical composition of cerebrospinal fluid (CSF) microdialysate from the rat brain. In vivo microdialysis techniques were used in several brain regions including the frontal cortex, amygdala, striatum, nucleus accumbens and third ventricle and dialysate samples (20microl) were subsequently analyzed by one and two-dimensional 1H NMR experiments using a Varian nanoprobe. Neurochemical resonances were assigned on the basis of published chemical shifts [Lindon et al., Ann. Rep. NMR Spectrosc. 38 (1999) 1-88], correlation experiments and addition of standard compounds. Glucose, lactate, formate, pyruvate, creatinine, gamma-hydroxybutyrate, acetate, glutamate, glycine, tyrosine, isoleucine, leucine, alanine and choline were some of the neurochemicals unambiguously assigned. Additional studies in the frontal cortex showed that amino acids such as glutamate, alanine and isoleucine were sensitive to local tetrodotoxin (TTX) infusion. The NMR spectra were also subjected to multivariate statistical methods to compare the different brain regions examined. To our knowledge, the present experiments are the first to describe the combination of nanoprobe NMR technology with in vivo microdialysis for the analysis of brain neurochemistry in freely-moving rats.

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.

The synthesis and biological evaluation of quinolyl-piperazinyl piperidines as potent serotonin 5-HT1A antagonists.

As part of an effort to identify 5-HT(1A) antagonists that did not possess typical arylalkylamine or keto/amido-alkyl aryl piperazine scaffolds, prototype compound 10a was identified from earlier work in a combined 5-HT(1A) antagonist/SSRI program. This quinolyl-piperazinyl piperidine analogue displayed potent, selective 5-HT(1A) antagonism but suffered from poor oxidative metabolic stability, resulting in low exposure following oral administration. SAR studies, driven primarily by in vitro liver microsomal stability assessment, identified compound 10b, which displayed improved oral bioavailability and lower intrinsic clearance. Further changes to the scaffold (e.g., 10r) resulted in a loss in potency. Compound 10b displayed cognitive enhancing effects in a number of animal models of learning and memory, enhanced the antidepressant-like effects of the SSRI fluoxetine, and reversed the sexual dysfunction induced by chronic fluoxetine treatment.

Mass‐Directed Normal‐Phase Preparative HPLC with Atmospheric Pressure Chemical Ionization Detection

Abstract A novel approach to auto‐purification of a wide variety of organic compounds is described. It is based on normal‐phase (NP) gradient high performance liquid chromatography (HPLC), performed on a 2 × 15 cm cyano column hyphenated with atmospheric pressure chemical ionization (APCI) source, and a single quad mass spectrometer (MS). A commercially available preparative HPLC–MS system, equipped with mass‐directed fraction collection capabilities, has been successfully used for NP purification of neutral, acidic, and basic pharmacologically active compounds. Samples of 10–100 mg were chromatographed in gradients of methanol in hydrophobic organic solvents, and collected using generic chromatographic, detection and fraction collection experimental parameters. “Smart” collection—one component–one collection vessel—with 90–95% recovery, was achieved by controlling injection volume and gradient slope and by adding acetic acid and diethylamine to the mobile phase to keep peak elution volumes below 50 mL. Auto‐collection of a solute was based on the main ion in its APCI–MS spectrum. The technique described has been also successfully used for chiral preparative HPLC applications and purification of non‐UV‐active compounds.

Developing a Simple NMR Interface: WWNMR

We have designed and implemented an automated walkup NMR computer application for use by synthetic chemists. The Wyeth Walkup NMR (WWNMR) application is a TCL/TK graphical user interface front-end to Varians NMR automation routines. The key feature of WWNMR is that the chemists are isolated from the spectrometers control program (VNMR) and only enter information to WWNMR windows for a sample to be run. The resulting data can be printed out on paper, or stored on disk for easy access by all users at their desktops. Proton and carbon NMR experiments are available. An overview of the WWNMR application will be presented.

An Automated Approach to NMR Data Handling for Compound Validation and Registration

We have designed and implemented a computer application called CHECKIN to submit open-access NMR data to the Wyeth Research compound validation and registration workflow process. At Wyeth Research, all analytical data used for compound validation and subsequent registration must be examined by Discovery Analytical Chemistry (DAC) staff. The traditional process required the medicinal chemist to submit vials of a pure sample to DAC to perform assays to validate its structure. For NMR analysis, DAC used this material to prepare a sample in deuterated solvent, which is used to collect one-dimensional 1H NMR data. DAC then reviews the spectra for consistency with the proposed chemical structure. This evaluation process was designed to reduce the number of misidentified compounds submitted to the corporate library by having a second set of eyes examine the data for each submission. This new computer application allows the medicinal chemist to select previously acquired hands-on NMR data and enter it into the standard workflow for evaluation, bypassing the sample preparation and data acquisition steps. The CHECKIN application saves time for the medicinal chemist and DAC staff, as well as compound library material for Wyeth. An explanation of the workflow and implementation for Bruker and Varian NMR data will be described.

Characterization of a methoxylated oxazol-5-one derivative: an unexpected by-product in a dehydropeptide synthesis

During the synthesis of the oxazol-5-one derivative (2) of t-butoxycarbonyl-glycyl-2,3-didehydrophenylalanine, the unexpected side product the 2-methoxyoxazol-5-one (3) was isolated and subsequently characterized by n.m.r., i.r., and single-crystal X-ray diffraction analysis.