HPLC-UV monitored photostability-test of LE404 and identification of the degradation products via NMR and LC‑HRMS

Abstract

Abstract Dibenzoazecines are a new class of potential neuroleptics with a high potential for the treatment of schizophrenia. Initial stress tests indicated that the lead compound, LE404, decomposed when exposed to oxygen and sunlight. In this follow up study, the influence of oxidative stress and photosensitivity was examined in compliance with ICH guidelines. The results of this study are important for identifying and predicting potential impurities of LE404 and for adjusting storage condition to prevent compound degradation. The exposure of LE404 to a light source, similar to the natural sunlight spectrum and intensity, was investigated in compliance with ICH-Q1B. The influence of oxidizing agents was investigated under exclusion of light. Two degradation products were observed. The extent and rate of degradation in both experiments were continuously monitored using RP-HPLC-UV. Chromatographic separations were achieved with a phenomenex™ Gemini 5 µm C18 110 Å (250×4.60 mm) column at 220 nm, using acetonitrile/KH2PO4 buffer (4 mmol⋅L-1, pH 2.5) as mobile phase. The photodegradation product was isolated by using semi-preparative RP-HPLC. The oxidation product was obtained by quantitative conversion of LE404 in hydrogen peroxide followed through purification using preparative TLC. The structures of both degradation products were elucidated using HR-MS/MS, 1D- and 2D-NMR as well as FTIR spectroscopy. The structural characterization of the degradation products serves as the basis for subsequent investigations into their toxicity. This is of high importance because phototoxic and photoallergic reactions pose a significant risk, especially with long-term medications, as it is not possible to completely avoid exposure to sunlight.