Milligram scale enantioresolution of promethazine and its main metabolites, determination of their absolute configuration and assessment of enantioselective effects on human SY-SY5Y cells

Abstract

The misuse of pharmaceuticals has significantly increased in recent decades, becoming a major public health concern. The risks associated with medication misuse are particularly high in cases of overdose, especially when the active substances are chiral, as enantioselectivity plays an important role in toxicity. Promethazine (PMZ) is a chiral antihistamine marketed as a racemate and it is misused in "Purple Drank", a recreational drug beverage, that combines codeine and/or PMZ, with soda or alcohol leading to serious health consequences and fatalities in consumers around the world, particularly among teenagers. Information regarding the enantioselectivity in the toxicity of (R,S)-PMZ and its main metabolites, namely promethazine sulfoxide (PMZSO) and desmonomethyl promethazine (DMPMZ), is unknown. This work reported, for the first time, the enantioseparation, in milligram scale, of (R,S)-PMZ, (R,S)-DMPMZ, (R,S)- PMZSO and the determination of their absolute configurations by electronic circular dichroism (ECD). The enantioseparation of all the six enantiomers was accomplished in a homemade semi-preparative column with amylose tris-3,5-dimethylphenylcarbamate (AD) coated with aminopropyl Nucleosil silica. The enantiomeric purity was evaluated using the analytical Lux® 3 µm i-Amylose-3 column, yielding enantiomeric purity values ranging between 94.4% and 99.7%. The elution order of all the enantiomers was accomplished combining the ECD results with an optical rotation detector. The elution order of the enantiomers was influenced only by the chiral selector, rather than the mobile phase. The cytotoxicity of the racemates and the isolated enantiomers towards differentiated SH-SY5Y cells was evaluated. (R,S)-DMPMZ exhibited a significantly higher cytotoxicity than (R,S)-PMZ, suggesting the metabolic bioactivation of (R,S)-PMZ. Conversely, no significant cytotoxicity was found for (R,S)-PMZSO, underscoring a metabolic detoxification pathway. Remarkably, enantioselectivity was observed for the cytotoxicity of PMZ; (R)-PMZ was significantly more cytotoxic than (S)-PMZ. The results underscore the importance to isolate the enantiomers in their enantiomerically form and their correct identification for toxicity enantioselectivity studies, which are vital to understand the drug's behaviour and safety, especially in case of overdoses.

Keywords: Absolute configuration; Enantioselectivity; Enantioseparation; Neurotoxicity; Promethazine; SH-SY5Y cells.