N-Benzylphenethylamines are novel psychedelic substances increasingly used for research, diagnostic, or recreational purposes. To date, only a few metabolism studies have been conducted for N-2-methoxybenzylated compounds (NBOMes). Thus, the available 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe) metabolism data are limited. Herein, we investigated the metabolic profile of 25CN-NBOMe in vivo in rats and in vitro in Cunninghamella elegans (C. elegans) mycelium and human liver microsomes. Phase I and phase II metabolites were first detected in an untargeted screening, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of the most abundant metabolites by comparison with in-house synthesized reference materials. The major metabolic pathways described within this study (mono- and bis-O-demethylation, hydroxylation at different positions, and combinations thereof, followed by the glucuronidation, sulfation, and/or N-acetylation of primary metabolites) generally correspond to the results of previously reported metabolism of several other NBOMes. The cyano functional group was either hydrolyzed to the respective amide or carboxylic acid or remained untouched. Differences between species should be taken into account in studies of the metabolism of novel substances.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 5 16 628 Prague Czech Republic

Department of Experimental Neurobiology National Institute of Mental Health Topolová 748 250 67 Klecany Czech Republic

Department of Organic Chemistry University of Chemistry and Technology Prague Technická 5 16 628 Prague Czech Republic

Forensic Laboratory of Biologically Active Substances Department of Chemistry of Natural Compounds University of Chemistry and Technology Prague Technická 5 16 628 Prague Czech Republic

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Glennon R.A., Dukat M., El-Bermawy M., Law H., Angeles J.D.L., Teitler M., King A., Herrick-Davis K. Influence of amine substituents on 5-HT2A versus 5-HT2C binding of phenylalkyl- and indolylalkylamines. J. Med. Chem. 1994;37:1929–1935. doi: 10.1021/jm00039a004. PubMed DOI

Ettrup A., Hansen M., Santini M.A., Paine J., Gillings N., Palner M., Lehel S., Herth M.M., Madsen J., Kristensen J., et al. Radiosynthesis and in vivo evaluation of a series of substituted 11 C-phenethylamines as 5-HT 2A agonist PET tracers. Eur. J. Nucl. Med. Mol. Imaging. 2011;38:681–693. doi: 10.1007/s00259-010-1686-8. PubMed DOI

Ettrup A., Da Cunha-Bang S., McMahon B., Lehel S., Dyssegaard A., Skibsted A.W., Jørgensen L.M., Hansen M., O Baandrup A., Bache S., et al. Serotonin 2A Receptor Agonist Binding in the Human Brain with [11C]Cimbi-36. Br. J. Pharmacol. 2014;34:1188–1196. doi: 10.1038/jcbfm.2014.68. PubMed DOI PMC

Hansen M. Ph.D. Thesis. University of Copenhagen; Copenhagen, Switzerland: 2010. Design and Synthesis of Selective Serotonin Receptor Agonists for Positron Emission Tomography Imaging of the Brain.

Hansen M., Phonekeo K., Paine J.S., Leth-Petersen S., Begtrup M. Synthesis and structure−activity relationships of N‑Benzyl phenethylamines as 5-HT2A/2C agonists. ACS Chem. Neurosci. 2014;5:243–249. doi: 10.1021/cn400216u. PubMed DOI PMC

Jensen A.A., McCorvy J.D., Leth-Petersen S., Bundgaard C., Liebscher G., Kenakin T.P., Bräuner-Osborne H., Kehler J., Kristensen J.L. Detailed characterization of the in vitro pharmacological and pharmacokinetic properties of N-(2-hydroxybenzyl)-2, 5-dimethoxy-4-cyanophenylethylamine (25CN-NBOH), a highly selective and brain-penetrant 5-HT2A receptor agonist. J. Pharmacol. Exp. Ther. 2017;361:441–453. doi: 10.1124/jpet.117.239905. PubMed DOI

Zuba D., Sekuła K., Buczek A. 25C-NBOMe—New potent hallucinogenic substance identified on the drug market. Forensic Sci. Int. 2013;227:7–14. doi: 10.1016/j.forsciint.2012.08.027. PubMed DOI

Poklis J.L., Raso S.A., Alford K.N., Poklis A., Peace M.R. Analysis of 25I-NBOMe, 25B-NBOMe, 25C-NBOMe and other dimethoxyphenyl-N-[(2-Methoxyphenyl)Methyl] ethanamine derivatives on blotter paper. J. Anal. Toxicol. 2015;39:617–623. doi: 10.1093/jat/bkv073. PubMed DOI PMC

Halberstadt A.L. Pharmacology and toxicology of N-benzylphenethylamine (“NBOMe”) hallucinogens. Curr. Top. Behav. Neurosci. 2017;32:283–311. doi: 10.1007/7854. PubMed DOI

Glennon R.A., Titeler M., McKenney J.D. Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents. Life Sci. 1984;35:2505–2511. doi: 10.1016/0024-3205(84)90436-3. PubMed DOI

Titeler M., Lyon R.A., Glennon R.A. Radioligand binding evidence implicates the brain 5-HT2 receptor as a site of action for LSD and phenylisopropylamine hallucinogens. Psychopharmacology. 1988;94:213–216. doi: 10.1007/BF00176847. PubMed DOI

Vollenweider F.X., Vollenweider-Scherpenhuyzen M.F.I., Bäbler A., Vogel H., Hell D. Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport. 1998;9:3897–3902. doi: 10.1097/00001756-199812010-00024. PubMed DOI

Quednow B.B., Kometer M., Geyer M.A., Vollenweider F.X. Psilocybin-induced deficits in automatic and controlled inhibition are attenuated by ketanserin in healthy human volunteers. Neuropsychopharmacology. 2012;37:630–640. doi: 10.1038/npp.2011.228. PubMed DOI PMC

Valle M., Maqueda A.E., Rabella M., Rodríguez-Pujadas A., Antonijoan R.M., Romero S., Alonso J.F., Mañanas M.À., Barker S., Friedlander P., et al. Inhibition of alpha oscillations through serotonin-2A receptor activation underlies the visual effects of ayahuasca in humans. Eur. Neuropsychopharmacol. 2016;26:1161–1175. doi: 10.1016/j.euroneuro.2016.03.012. PubMed DOI

Halberstadt A.L. Recent advances in the neuropsychopharmacology of serotonergic hallucinogens. Behav. Brain Res. 2014;277:99–120. doi: 10.1016/j.bbr.2014.07.016. PubMed DOI PMC

Pertz H.H., Heim R., Elz S. N-Benzylated phenylethanamines are highly potent partial agonists at 5-HT2A receptors (abstract) Arch. Pharm. Pharm. Med. Chem. 2000;333:30.

Braden M.R., Parrish J.C., Naylor J.C., Nichols D.E. Molecular interaction of serotonin 5-HT 2A receptor residues phenethylamine agonists. Mol. Pharmacol. 2006;70:1956–1964. doi: 10.1124/mol.106.028720. PubMed DOI

Eshleman A.J., Wolfrum K.M., Reed J.F., Kim S.O., Johnson R.A., Janowsky A. Neurochemical pharmacology of psychoactive substituted N-benzylphenethylamines: High potency agonists at 5-HT2A receptors. Biochem Pharmacol. 2018;158:27–34. doi: 10.1016/j.bcp.2018.09.024. PubMed DOI PMC

Heim R. Ph.D. Thesis. Fachbereich Biologie, Chemie Pharmazie der Freien Universität; Berlin, Germany: Mar 25, 2003. Synthese und Pharmakologie Potenter 5-HT2A-Rezeptoragonisten mit N-2-Methoxybenzyl-Partialstruktur.

Elmore J.S., Decker A.M., Sulima A., Rice K.C., Partilla J.S., Blough B.E., Baumann M.H., Carolina N., Section S. Comparative neuropharmacology of N-(2-methoxybenzyl)-2,5-dimethoxyphenethylamine (NBOMe) hallucinogens and their 2C counterparts in male rats. Neuropharmacology. 2019;142:240–250. doi: 10.1016/j.neuropharm.2018.02.033. PubMed DOI PMC

Rickli A., Luethi D., Reinisch J., Buchy D., Hoener M.C., Liechti M.E. Receptor interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy-substituted phenethylamines (2C drugs) Neuropharmacology. 2015;99:546–553. doi: 10.1016/j.neuropharm.2015.08.034. PubMed DOI

Halberstadt A.L., Geyer M.A. Characterization of the head-twitch response induced by hallucinogens in mice. Detection of the behavior based on the dynamics of head movement. Psychopharmacology. 2013;227:727–739. doi: 10.1007/s00213-013-3006-z. PubMed DOI PMC

Canal C.E., Morgan D. Head-twitch response in rodents induced by the hallucinogen 2,5-dimethoxy-4-iodoamphetamine: A comprehensive history, a re-evaluation of mechanisms, and its utility as a model. Drug Test. Anal. 2012;4:556–576. doi: 10.1002/dta.1333. PubMed DOI PMC

Halberstadt A.L., Chatha M., Klein A.K., Wallach J., Brandt S.D. Correlation between the potency of hallucinogens in the mouse head-twitch response assay and their behavioral and subjective effects in other species. Neuropharmacology. 2020;167:107933. doi: 10.1016/j.neuropharm.2019.107933. PubMed DOI PMC

Ettrup A., Holm S., Hansen M., Wasim M., Santini M.A., Palner M., Madsen J., Svarer C., Kristensen J.L., Knudsen G.M. Preclinical safety assessment of the 5-HT2A receptor agonist PET radioligand [11C]Cimbi-36. Mol. Imaging Biol. 2013;15:376–383. doi: 10.1007/s11307-012-0609-4. PubMed DOI

Halberstadt A.L., Geyer M.A. Effects of the hallucinogen 2,5-dimethoxy-4-iodophenethylamine (2C-I) and superpotent N-benzyl derivatives on the head twitch response. Neuropharmacology. 2014;77:200–207. doi: 10.1016/j.neuropharm.2013.08.025. PubMed DOI PMC

Suzuki J., Poklis J.L., Poklis A. “My friend said it was good LSD”: A suicide attempt following analytically confirmed 25I-NBOMe ingestion. J. Psychoact. Drugs. 2014;46:37–41. doi: 10.1080/02791072.2014.960111. PubMed DOI PMC

Poklis J.L., Devers K.G., Arbefeville E.F., Pearson J.M., Houston E., Poklis A. Postmortem detection of 25I-NBOMe [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine] in fluids and tissues determined by high performance liquid chromatography with tandem mass spectrometry from a traumatic death. Forensic Sci. Int. 2013;234:e14–e20. doi: 10.1016/j.forsciint.2013.10.015. PubMed DOI PMC

Poklis J.L., Dempsey S.K., Liu K., Ritter J.K., Wolf C., Zhang S., Poklis A. Identification of metabolite biomarkers of the designer hallucinogen 25I-NBOMe in mouse hepatic microsomal preparations and human urine samples associated with clinical intoxication. J. Anal. Toxicol. 2015;39:607–616. doi: 10.1093/jat/bkv079. PubMed DOI PMC

Wohlfarth A., Roman M., Andersson M., Kugelberg F.C., Diao X., Carlier J., Eriksson C., Wu X., Konradsson P., Josefsson M., et al. 25C-NBOMe and 25I-NBOMe metabolite studies in human hepatocytes, in vivo mouse and human urine with high-resolution mass spectrometry. Drug Test. Anal. 2017;9:680–698. doi: 10.1002/dta.2044. PubMed DOI

Caspar A.T., Helfer A.G., Michely J.A., Auwärter V., Brandt S.D., Meyer M.R., Maurer H.H. Studies on the metabolism and toxicological detection of the new in psychoactive designer drug 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) human and rat urine using GC-MS, LC-MS n, and LC-HR-MS/MS. Anal. Bioanal. Chem. 2015;407:6697–6719. doi: 10.1007/s00216-015-8828-6. PubMed DOI

Caspar A.T., Brandt S.D., Stoever A.E., Meyer M.R., Maurer H.H. Metabolic fate and detectability of the new psychoactive substances 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25B-NBOMe) and 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) in human and rat urine by GC-MS, LC-MS n, and LC-HR-MS/MS approaches. J. Pharm. Biomed. Anal. 2017;134:158–169. doi: 10.1016/j.jpba.2016.11.040. PubMed DOI

Nielsen L.M., Holm B., Leth-Petersen S., Kristensen J.L., Linnet K. Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH. Drug Test Anal. 2016;9:671–679. doi: 10.1002/dta.2031. PubMed DOI

Boumrah Y., Humbert L., Phanithavong M. In vitro characterization of potential CYP- and UGT-derived metabolites of the psychoactive drug 25B-NBOMe using LC-high resolution MS. Drug Test Anal. 2016;8:248–256. doi: 10.1002/dta.1865. PubMed DOI

Leth-Petersen S., Gabel-jensen C., Gillings N., Lehel S., Hansen H.D., Knudsen G.M., Kristensen J.L. Metabolic fate of hallucinogenic NBOMes. Chem. Res. Toxicol. 2016;26:96–100. doi: 10.1021/acs.chemrestox.5b00450. PubMed DOI

Seo H., Kim I.S., Kim Y., Yoo H.H., Hong J. Metabolic profile determination of 25N-NBOMe in human liver microsomes by liquid chromatography-quadrupole time-of-flight mass spectrometry. Int. J. Legal Med. 2019;133:833–841. doi: 10.1007/s00414-018-1904-7. PubMed DOI

Caspar A.T., Meyer M.R., Westphal F., Weber A.A., Maurer H.H. Nano liquid chromatography-high-resolution mass spectrometry for the identification of metabolites of the two new psychoactive substances N-(ortho-methoxybenzyl)-3,4-dimethoxyamphetamine and N-(ortho-methoxybenzyl)-4-methylmethamphetamine. Talanta. 2018;188:111–123. doi: 10.1016/j.talanta.2018.05.064. PubMed DOI

Grafinger K.E., Stahl K., Wilke A., König S., Weinmann W. In vitro phase I metabolism of three phenethylamines 25D-NBOMe, 25E-NBOMe and 25N-NBOMe using microsomal and microbial models. Drug Test Anal. 2018;10:1607–1626. doi: 10.1002/dta.2446. PubMed DOI

Ketha H., Webb M., Clayton L., Li S. Gas Chromatography Mass Spectrometry (GC-MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine. Curr. Protoc. Toxicol. 2017;74:4.43.1–4.43.10. doi: 10.1002/cptx.33. PubMed DOI

Richter L.H.J., Maurer H.H., Meyer M.R. New psychoactive substances. Studies on the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam using human liver preparations in comparison to primary human hepatocytes, and human urine. Toxicol. Lett. 2017;280:142–150. doi: 10.1016/j.toxlet.2017.07.901. PubMed DOI

Temporal K.H., Scott K.S., Mohr A.L.A., Logan B.K. Metabolic profile determination of NBOMe compounds using human liver microsomes and comparison with findings in authentic human blood and urine. J. Anal. Toxicol. 2017;41:646–657. doi: 10.1093/jat/bkx029. PubMed DOI

Caspar A.A.T., Meyer M.R., Maurer H.H. Human cytochrome P450 kinetic studies on six N-2-methoxybenzyl (NBOMe)-derived new psychoactive substances using the substrate depletion approach. Toxicol. Lett. 2017;285:1–8. doi: 10.1016/j.toxlet.2017.12.017. PubMed DOI

Leth-Petersen S., Bundgaard C., Hansen M., Carnerup M.A., Kehler J., Kristensen J.L. Correlating the metabolic stability of psychedelic 5-HT 2A agonists with anecdotal reports of human oral bioavailability. Neurochem. Res. 2014;39:2018–2023. doi: 10.1007/s11064-014-1253-y. PubMed DOI

Campbell J.L., Collings B.A., Le Blanc J.C.Y., Hager J.W. A novel MS3 experiment for quantifying ions with a linear ion trap. Can. J. Chem. 2018;96:653–663. doi: 10.1139/cjc-2017-0734. DOI

Markus B., Kwon C. In vitro metabolism of aromatic nitriles. J. Pharm. Sci. 1994;83:1729–1734. doi: 10.1002/jps.2600831216. PubMed DOI

ThermoFisher. [(accessed on 17 March 2021)]; Available online: https://www.thermofisher.com/cz/en/home/references/protocols/drug-discovery/adme-tox-protocols/microsomes-protocol.html.

Chemistry Archive. [(accessed on 4 January 2021)]; Available online: https://erowid.org/archive/rhodium/chemistry/edda.html.

Butterick J.R., Unrau A.M. Reduction of β-nitrostyrene with sodium bis-(2-methoxyethoxy)-aluminium dihydride. A convenient route to substituted phenylisopropylamines. J. Chem. Soc. Chem. Commun. 1974;8:307–308. doi: 10.1039/C39740000307. DOI

Xu Y.-Z., Chen C. Synthesis of deuterium labeled phenethylamine derivatives. J. Label Compd. Radiopharm. 2006;49:1187–2000. doi: 10.1002/jlcr.1139. DOI

Cheng A.C., Castagnoli N. Synthesis and physicochemical and neurotoxicity studies of l-(4-substituted-2,5-dihydroxyphenyl)-2-aminoethane analogues of 6-hydroxydopamine. J. Med. Chem. 1984;27:513–520. doi: 10.1021/jm00370a014. PubMed DOI

Hájková K., Jurásek B., Čejka J., Štefková K., Páleníček T., Sýkora D., Kuchař M. Synthesis and identification of deschloroketamine metabolites in rats’ urine and a quantification method for deschloroketamine and metabolites in rats’ serum and brain tissue using liquid chromatography tandem mass spectrometry. Drug Test. Anal. 2020;12:343–360. doi: 10.1002/dta.2726. PubMed DOI

2C-B-Fly-NBOMe Metabolites in Rat Urine, Human Liver Microsomes and C. elegans: Confirmation with Synthesized Analytical Standards