Oliver McConnell

High throughput artificial membrane permeability assay for blood-brain barrier.

The recent advances in high throughput screening for biological activities and combinatorial chemistry have greatly expanded the number of drug candidates. Rapid screening for BBB penetration potential early in drug discovery programs provides important information for compound selection and guidance of synthesis for desirable CNS properties. In this paper, we discuss a modification of the parallel artificial membrane permeation assay (PAMPA) for the prediction of blood-brain barrier penetration (PAMPA-BBB). The assay was developed with 30 structurally diverse commercial drugs and validated with 14 Wyeth Research compounds. The PAMPA-BBB assay has the advantages of: predicting passive blood-brain barrier penetration with high success, high throughput, low cost, and reproducibility.

Pharmaceutical profiling method for lipophilicity and integrity using liquid chromatography-mass spectrometry

A method is described for the simultaneous profiling of sample lipophilicity, integrity, and purity. The method is rapid and is applicable to high throughput profiling of pharmaceutical properties in drug discovery. A short Polaris C(18) column is used with a rapid, wide-polarity mobile phase gradient, UV detection, and MS analysis. The lipophilicity of each component is estimated from a calibration curve using six drug or organic compounds and plotting their respective measured retention time versus LogD(7.4) (literature). The correlation of LogD(7.4) (literature) to LogD(7.4) (HPLC) for 60 structurally diverse drugs has a correlation coefficient r(2) of 0.89. The method is applicable to compounds with MW>200 and retention time>1.5 min for rapid, initial pharmaceutical profiling in drug discovery.

Relative hydrophobicity and lipophilicity of drugs measured by aqueous two-phase partitioning, octanol-buffer partitioning and HPLC. A simple model for predicting blood-brain distribution.

Relative hydrophobicity and lipophilicity of 63 compounds with known permeability through the blood-brain barrier (BBB) was examined by partitioning in aqueous dextran-poly(ethylene glycol) two-phase system and octanol-buffer system, and by gradient RP-HPLC at pH 7.4. Combination of the relative hydrophobicity estimates, N(CH(2)) obtained by aqueous two-phase partitioning and the lipophilicity (logD(exp) or logD(HPLC)) values obtained by the shake-flask technique or HPLC technique allows one to differentiate between compounds capable of crossing the BBB and those that cannot. A simple model for predicting blood-brain distribution is proposed.

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.

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.

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.

NMR CHARACTERIZATION OF AN S-LINKED GLUCURONIDE METABOLITE OF THE POTENT, NOVEL, NONSTEROIDAL PROGESTERONE AGONIST TANAPROGET

Tanaproget is a first-in-class nonsteroidal progesterone receptor agonist that is being investigated for use in contraception. A major in vitro and in vivo metabolite of tanaproget formed in humans was initially characterized as a glucuronide of tanaproget. However, whether the glucuronide was linked to the nitrogen or sulfur of the benzoxazine-2-thione group in tanaproget could not be determined by liquid chromatography/mass spectrometry (LC/MS) and LC-tandem mass spectrometry analysis. To obtain additional structural details for this metabolite, additional quantities were generated from rat liver microsomal incubations and purified by high-performance liquid chromatography (HPLC) for NMR analysis. The NMR data for the metabolite confirmed that the glucuronide was covalently bound to either the sulfur or the nitrogen of the benzoxazine-2-thione moiety. The lack of key through-bond (scalar) and through-space (dipolar) one-dimensional (1D) and two-dimensional (2D) NMR couplings and correlations in the metabolite spectra (due primarily to low sample concentration) precluded an unambiguous structure elucidation. Subsequent synthesis of the S- and N-glucuronides of tanaproget from tanaproget facilitated the unambiguous regio- and stereochemical assignment of the metabolite by comparison of 1D NMR chemical shifts and scalar coupling constants, 2D NMR correlations, and HPLC and LC/MS characteristics between the synthetic compounds and the metabolite. From extensive comparison of the spectral and chromatographic data of the microsomally derived metabolite and the synthetic compounds, the metabolite has been determined to be the S-(β)-d-glucuronide of tanaproget.