Preparative Microfluidic Electrosynthesis of Drug Metabolites

Abstract

In vivo, a drug molecule undergoes its first chemical transformation within the liver via CYP450-catalyzed oxidation. The chemical outcome of the first pass hepatic oxidation is key information to any drug development process. Electrochemistry can be used to simulate CYP450 oxidation, yet it is often confined to the analytical scale, hampering product isolation and full characterization. In an effort to replicate hepatic oxidations, while retaining high throughput at the preparative scale, microfluidic technology and electrochemistry are combined in this study by using a microfluidic electrochemical cell. Several commercial drugs were subjected to continuous-flow electrolysis. They were chosen for their various chemical reactivity: their metabolites in vivo are generated via aromatic hydroxylation, alkyl oxidation, glutathione conjugation, or sulfoxidation. It is demonstrated that such metabolites can be synthesized by flow electrolysis at the 10 to 100 mg scale, and the purified products are fully characterized. KEYWORDS: Drug metabolites, electrochemistry, microfluidic synthesis, continuous-flow oxidation T he first step toward elimination of xenobiotic compounds in vivo occurs predominantly through first pass hepatic oxidation. In the liver, the oxidation takes place at the iron center of the porphyrin that constitutes the catalytic site of the CYP450 family of enzymes. 1,2 The oxidized metabolites (Phase I) may undergo subsequent transformations (Phase II) including, among others, conjugation with glutathione (GSH). This is of particular importance in the design of new drug candidates since their toxicity levels are regulated by such metabolic processes. 3,4 Significant research effort has been devoted to develop synthetic methodologies to simulate the metabolism of drugs. These methodologies fall into four distinct categories: microsomal incubation, porphyrin-catalyzed chemical oxidation, Fenton-type reactions, and electrochemical oxidations. 5,6 Electrosynthetic transformations offer the advantage of atom economy since the oxidation takes place at the surface of an electrode, which provides the electron from a current source. 7 These transformations are thus heterogeneous reactions by definition. To achieve high conversion, the substrate solution must be conductive, and the ratio of electrode surface-tosolution volume ought to be as high as possible. While electrosynthesis has been extensively performed in batch mode, 8,9 continuous-flow technology provides increased electrode surface ratios by design. 9 Additionally, at the microfluidic scale the distance between the working electrode and the counter electrode is shortened. This can help overcome conversion limitations linked to solution resistivity, and even enable electrolyte-free reactions. 10 In the past few years, the study of drug metabolites has benefited from the emergence of in-line electrochemical/mass spectrometry (EC-MS) systems used to generate phase I and phase II metabolites. 13,14 Unfortunately, full structure elucidation is limi...

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