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.

Keywords: Fentanyl analogs; Human hepatocytes; LC-QTOF-MS; Metabolism.

Fig. 1

Proposed metabolic pathway of cyclopropylfentanyl (A, top) with the modifications highlighted in blue and major metabolites underlined. Markush structures are used to indicate when several positions of the modifications are possible with the exceptions of A2 and A5 where modifications are drawn as one possible isomer. Extracted ion chromatograms of cyclopropylfentanyl and its corresponding metabolites formed after 5 h of incubation with human hepatocytes are also depicted (bottom) with close-ups of the minor metabolites A2, A3 and A5. (Color figure online)

Fig. 2

Proposed metabolic pathway of cyclobutylfentanyl (B, top) with the modifications highlighted in blue and major metabolites underlined. Markush structures are used to indicate when several positions of the modifications are possible. Extracted ion chromatograms of cyclobutylfentanyl and its corresponding metabolites formed after 5 h of incubation with human hepatocytes are also depicted (bottom). (Color figure online)

Fig. 3

Proposed metabolic pathway of cyclopentylfentanyl (C, top) with the modifications highlighted in blue and major metabolites underlined. Markush structures are used to indicate when several positions of the modifications are possible. Extracted ion chromatograms of cyclopentylfentanyl and its corresponding metabolites formed after 5 h of incubation with human hepatocytes are also depicted (bottom). (Color figure online)

Fig. 4

Proposed metabolic pathway of cyclohexylfentanyl (D, top) with the modifications highlighted in blue and major metabolites underlined. Markush structures are used to indicate when several positions of the modifications are possible. Extracted ion chromatograms of cyclohexylfentanyl and its corresponding metabolites formed after 5 h of incubation with human hepatocytes are also depicted (bottom). (Color figure online)

Fig. 5

Proposed metabolic pathway of 2,2,3,3-tetramethylcyclopropylfentanyl (TMCPF, E, top) with the modifications highlighted in blue and major metabolites underlined. Markush structures are used to indicate when several positions of the modifications are possible with the exceptions of E6, E7 and E10 where modifications are drawn as one possible isomer. Extracted ion chromatograms of TMCPF and its corresponding metabolites formed after 5 h of incubation with human hepatocytes are also depicted (bottom). (Color figure online)

Fig. 6

Percentages, by peak area, of normetabolites (black), metabolites oxidized on the piperidine moiety (dark gray), amide hydrolysis products (light gray) and metabolites oxidized on the aliphatic ring (white) relative to the total area of all metabolites after 1 h incubation for each fentanyl analog. Metabolites belonging to more than one group, due to multiple biotransformations or ambiguity in structure assignment, were counted in both categories. The curves illustrate the increasing/decreasing trends of metabolites oxidized on the aliphatic ring (dashed) and normetabolites (solid), as the ring sizes increases

Fig. 7

MSMS fragmentation of the alicyclic fentanyl analogs. Diagnostic fragment ions are labeled a–g, linked to their respective substructure and displayed with their corresponding m/z values. Fragments for which the m/z vary with the alicyclic ring have been marked with a subscript X and where applicable the m/z is given for each alicyclic fentanyl analog. (Color figure online)