Tentative identification of the metabolites of (1-(cyclohexylmethyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone, and the product of its thermal degradation, by in vitro and in vivo methods
Synthetic cannabinoids (SCs), mimicking the psychoactive effects of cannabis, consist of a vast array of structurally diverse compounds. A novel compound belonging to the SC family, (1-(cyclohexylmethyl)-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (named TMCP-CHM in this article) contains a cyclopropane ring that isomerizes during the smoking process, resulting in a ring-opened thermal degradant with a terminal double bond in its structure. Metabolites of TMCP-CHM were tentatively identified in vitro (after incubation of the parent substance with S9 pooled human liver fraction) and in vivo (rat experimental model) studies by accurate-mass liquid chromatography–tandem mass spectrometry (LC–MS/MS). For the identification of the degradant metabolites, and to study biotransformation of parent substance in the human, urine and hair samples from patients, who had ingested the compound and were subsequently admitted to hospital with drug intoxications, were analyzed. Products of mono-, di-, trihydroxylation, carboxylation, and carboxylation combined with hydroxylation of TMCP-CHM and its degradant were detected in human urine. Metabolism of the degradant included addition of water to the terminal double bond followed by dehydration and formation of a cyclic metabolite. Degradant metabolites prevailed in comparison with metabolites of the parent substance in each metabolite group examined, except carboxylation. N-Dealkylated metabolites found in human urine originated only from the degradant. Most of the hydroxy metabolites were detected in human urine in both the free form and as glucuronides. The detection of monohydroxylated (M1.1-M1.3, M/A1.10) and carboxylated/hydroxylated (M4.2, M/A4.3) metabolites of TMCP-CHM and the hydrated form of the monohydroxylated metabolite of the degradant was found to be convenient for routine analysis.
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1H NMR spectrum of TMCP-CHM
Figure S2. 1H NMR spectrum of TMCP-CHM (expansions)
Figure S3. 13C NMR spectrum of TMCP-CHM
Figure S4. GC-MS spectra of TMCP-CHM (a) and TMCP-CHM degradant (b)
Figure S5. Proposed scheme of fragmentation pathways of the TMCP-CHM and its degradant (GC-MS, EI)
Figure S6. LC-QTOF extracted ion chromatograms of the monohydroxylated metabolites of TMCP-CHM and its thermal degradant (MS2, mass shift < 5 mDa). Ion transitions are shown on the right.
Table S1, LC-QTOF features of TMCP-CHM in vitro mono- and dihydroxylated metabolites
Figure S7. Dehydration of metabolites monohydroxylated on TMCP moiety. LC-QTOF extracted ion chromatograms (in vitro model, MS1, mass shift < 5 mDa) (a), (b). Mass spectrum of V1.5 (c). Presumable structures of monohydroxylated metabolites and artificial dehydrated forms (d). Metabolites detected only in vitro are denoted as ‘V’
Figure S8. LC-QTOF spectra of monohydroxylated metabolites originated from TMCP-CHM degradant (M/A1.9), hydrated form (M/A1.10) and dihydrofuran form (M/A1.12)
Figure S9. LC-QTOF (MS2, mass shift < 5 mDa) extracted ion chromatograms of the dihydroxylated metabolites of TMCP-CHM and its thermal degradant. Ion transitions are shown on the right. Metabolites detected only in vitro are denoted as ‘V’
Figure S10. Dehydration of dihydroxylated metabolites. LC-QTOF (in vitro model, MS1, mass shift < 5 mDa) extracted ion chromatograms (a), (b). Mass spectra of V2.8 (c) and V2.9 (d). Presumable structures of dihydroxylated metabolites and artificial dehydrated forms (e). Metabolites detected only in vitro are denoted as ‘V’.
Figure S11. LC-QTOF (MS2, mass shift < 5 mDa) extracted ion chromatograms of TMCP-CHM metabolites (carboxylated group). Ion transitions are shown on the right.
Figure S12. LC-QTOF (MS1, mass shift < 5 mDa) extracted ion chromatograms of TMCP-CHM, its thermal degradant and their dealkylated metabolites. Values of ions m/z are shown on the right.
Figure S13. LC-QTOF (MS2, mass shift < 5 mDa) extracted ion chromatograms (EICs) of TMCP-CHM metabolites (dealkylated with hydroxylation group). Ion transitions are shown on the right.
Figure S14. LC-QTOF (MS2, mass shift < 5 mDa) extracted ion chromatograms (EICs) of TMCP-CHM metabolites (dealkylated with dihydroxylation group). Ion transitions are shown on the right.
Figure S15. LC-QTOF spectra of dealkylated metabolites
Table S2. LC-QTOF features of common N-dealkylated metabolites originating from TMCP-CHM (rat model) and XLR-11 (human urine)
Figure S16. LC-QTOF (MS2, mass shift < 5 mDa) extracted ion chromatograms (EICs) of TMCP-CHM metabolites (trihydroxylation, M8 group) (a). Ion transitions are shown on the right. LC-QTOF spectra of trihydroxylated metabolites (b)
Table S3. LC-QTOF features of trihydroxylated metabolites of TMCP-CHM
Figure S17. Metabolites of M8 group (trihydroxylation) in the in vitro model. Dehydrated forms of trihydroxylated metabolites LC-QTOF (in vitro model, MS2, mass shift < 5 mDa) extracted ion chromatograms (a). Ion transitions are shown on the right. LC-QTOF spectra of V8.1 (b) and V8.3 (c).
Figure S18. Metabolites of M/A9 group (hydration with oxidation to carboxyl and additional hydroxylation) in human urine. LC-QTOF (in vitro model, MS2, mass shift < 5 mDa) extracted ion chromatograms (a). Ion transitions are shown on the right. LC-QTOF spectra of metabolites M/A9 group (b).
Table S4. LC-QTOF features of hydrated with oxidation to carboxyl and additional hydroxylation metabolites of TMCP-CHM degradant
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