Direct observation of a fluorinated anticonvulsant in brain tissue using 19F-NMR techniques

Abstract

A fluorinated derivative of an anticonvulsant γ-butyrolactone [α-(1,1-difluoroethyl)-α-methyl-γ-butyrolactone; γ-DFGBL] was synthesized as a probe for NMR spectroscopic observation of the drug in brain tissue. The fluorinated compound is an efficacious anticonvulsant in mice, and inhibits the specific binding of [ 35S] t-butylbicyclophosphorothionate ([ 35SITBPS) to mouse brain membranes with a concentration dependence similar to that of the non-fluorinated compound α-ethyl-α-methyl-γ-butyrolactone. Quantitative 19F-NMR spectroscopic studies, coupled with Chromatographic measurements of drug tissue concentration, showed that virtually all of the α-DFGBL in brain was NMR-observable and that, following intraperitoneal injection, α-DFGBL rapidly achieved millimolar concentrations in brain. The 19F-NMR spectra of a α-DFGBL in brain and liver tissue were broad (1–2 ppm) and complex, exhibiting multiple chemical shift features. The major chemical shift features in these spectra were assigned on the basis of differential extraction and comparison of 19F spin-spin relaxation times (T 2s) and 19F chemical shifts of α-DFGBL in tissue to those in pure solvents. The major feature at 10.4 ppm in the tissue spectra was assigned to a weakly polar, membrane-associated environment for the fluorinated compound, while the feature at 11.2 ppm was assigned to an aqueous environment for α-DFGBL. The drug was in slow exchange between these two environments in brain. In addition, the feature at lowest field (9.7–9.8 ppm) was identified as a water-soluble hydroxy-acid metabolite of α-DFGBL produced by the liver. These data indicate that γ-butyrolactone anticonvulsants achieve high concentrations in brain, where they exist in several, largely membrane-associated, environments. These findings are consistent with the purported action of the γ-butyrolactones as low-affinity modulators of γ-aminobutyric acid-A channels.