Correlations between metabolism and structural elements of the alicyclic fentanyl analogs cyclopropyl fentanyl, cyclobutyl fentanyl, cyclopentyl fentanyl, cyclohexyl fentanyl and 2,2,3,3-tetramethylcyclopropyl fentanyl studied by human hepatocytes and LC-QTOF-MS

Abstract

Recently, a number of fentanyl analogs have been implicated in overdose deaths in Europe and in the US. So far, little is known of the molecular behavior of the structurally related subgroup; the alicyclic fentanyls. In this study, reference standards of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2,2,3,3-tetramethylcyclopropyl fentanyl (TMCPF) at a final concentration of 5 µM were incubated with cryopreserved human hepatocytes (1 × 106 cells/mL) for 0, 1, 3 and 5 h. The metabolites formed were identified by liquid chromatography–quadrupole time-of-flight mass spectrometry analysis. The most abundant biotransformation found was N-dealkylation (formation of normetabolites) and oxidation of the alicyclic rings. As ring size increased, the significance of N-dealkylation decreased in favor of alicyclic ring oxidation. An example of this was cyclopropyl fentanyl, with a three-carbon ring, whose normetabolite covered 82% of the total metabolic peak area and no oxidation of the alicyclic ring was observed. In contrast, TMCPF, with a seven-carbon ring structure, rendered as much as 85% of its metabolites oxidized on the alicyclic ring. Other biotransformations found included oxidation of the piperidine ethyl moiety and/or the phenethyl substructure, glucuronidation as well as amide hydrolysis to form metabolites identical to despropionyl fentanyl. Taken together, this study provides a base for understanding the metabolism of a number of structurally related fentanyl analogs formed upon intake.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2330-9) contains supplementary material, which is available to authorized users.

In vitro metabolism of the novel synthetic opioid agonist cyclopropylfentanyl and subsequent confirmation in authentic human samples using liquid chromatography‐high resolution mass spectrometry

“…In a study reported by Åstrand et al, seven metabolites of CycP‐F were identified in in vitro incubations with cryopreserved human hepatocytes . Four of these metabolites were comparable to those reported here, with the major metabolite in both studies represented by the N ‐dealkylated metabolite.…”

“…Since carrying out this work, a study investigating the correlations between metabolism and structural elements of alicyclic fentanyl analogues, including CycP-F, has been reported. 28 Seven metabolites of CycP-F were identified utilising cryopreserved human hepatocytes in vitro, with good correlation to the metabolites observed in this study.…”

“…It has been suggested that extended incubation times and the required oxidative conditions can result in the increased production of these metabolites in vitro . However, Åstrand et al identified one N‐ oxide metabolite of CycP‐F which was observed after one hour of incubation, with no further N‐ oxide metabolites observed after three or five hours . Meyer et al .…”

“…Metabolites were identified in a two‐step investigative process; first with FS accurate mass analysis of the in vitro incubate and data mining for postulated metabolites, followed by targeted MS 2 analysis for any metabolites identified. Examination of the metabolism of fentanyl and other fentanyl‐analogues allowed the postulation of potential metabolites of CycP‐F . The possible pathways included N ‐dealkylation, amide hydrolysis, hydroxylation and potential sequential transformations.…”

“…Table 1 with no further N-oxide metabolites observed after three or five hours. 28 Meyer et al detected an N-oxide metabolite of isofentanyl in rat urine, but it was detected to much a greater extent in vitro. 33 Steuer et al detected two N-oxide metabolites of butyrfentanyl following in vitro incubations, however these were not detected in analysis of post-mortem blood or urine samples.…”