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Structural Elucidation of Metabolites of Synthetic Cannabinoid UR-144 by Cunninghamella elegans Using Nuclear Magnetic Resonance (NMR) Spectroscopy

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Abstract

The number of new psychoactive substances keeps on rising despite the controlling efforts by law enforcement. Although metabolism of the newly emerging drugs is continuously studied to keep up with the new additions, the exact structures of the metabolites are often not identified due to the insufficient sample quantities for techniques such as nuclear magnetic resonance (NMR) spectroscopy. The aim of the study was to characterise several metabolites of the synthetic cannabinoid (1-pentyl-1H-indol-3-yl) (2,2,3,3-tetramethylcyclopropyl) methanone (UR-144) by NMR spectroscopy after the incubation with the fungus Cunninghamella elegans. UR-144 was incubated with C. elegans for 72 h, and the resulting metabolites were chromatographically separated. Six fractions were collected and analysed by NMR spectroscopy. UR-144 was also incubated with human liver microsomes (HLM), and the liquid chromatography-high resolution mass spectrometry analysis was performed on the HLM metabolites with the characterised fungal metabolites as reference standards. Ten metabolites were characterised by NMR analysis including dihydroxy metabolites, carboxy and hydroxy metabolites, a hydroxy and ketone metabolite, and a carboxy and ketone metabolite. Of these metabolites, dihydroxy metabolite, carboxy and hydroxy metabolites, and a hydroxy and ketone metabolite were identified in HLM incubation. The results indicate that the fungus is capable of producing human-relevant metabolites including the exact isomers. The capacity of the fungus C. elegans to allow for NMR structural characterisation by enabling production of large amounts of metabolites makes it an ideal model to complement metabolism studies.

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Abbreviations

C. elegans :

Cunninghamella elegans

ESI :

Electrospray ionisation source

gCOSY :

Gradient correlation spectroscopy

gHMBC :

Gradient heteronuclear multiple bond correlation

gHSQC :

Gradient heteronuclear single quantum correlation

HLM :

Human liver microsomes

HPLC :

High-performance liquid chromatography

LC-QTOF-MS :

Liquid chromatography-quadrupole time-of-flight mass spectrometry

MS :

Mass spectrometry

NOESY :

Nuclear Overhauser effect spectroscopy

NMR :

Nuclear magnetic resonance

NPS :

New psychoactive substances

TMCP :

Tetramethylcyclopropyl

UR-144 :

(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone

References

  1. European Monitoring Centre for Drugs and Drug Addiction. European Drug Report. Trends and developments. Luxembourg: Publications Office of the European Union; 2017. p. 2017.

    Google Scholar 

  2. Carlier J, Diao X, Sempio C, Huestis MA. Identification of new synthetic cannabinoid ADB-CHMINACA (MAB-CHMINACA) metabolites in human hepatocytes. AAPS J. 2017;19:1–10. https://doi.org/10.1208/s12248-016-0037-5.

    Article  Google Scholar 

  3. Grafinger KE, Hädener M, König S, Weinmann W. Study of the in vitro and in vivo metabolism of the tryptamine 5-MeO-MiPT using human liver microsomes and real case samples. Drug Test Anal. in press; https://doi.org/10.1002/dta.2245.

  4. Meyer MR. New psychoactive substances: an overview on recent publications on their toxicodynamics and toxicokinetics. Arch Toxicol. 2016;90(10):2421–44. https://doi.org/10.1007/s00204-016-1812-x.

    Article  CAS  PubMed  Google Scholar 

  5. Watanabe S, Vikingsson S, Roman M, Green H, Kronstrand R, Wohlfarth A. In vitro and in vivo metabolite identification studies for the new synthetic opioids acetylfentanyl, acrylfentanyl, furanylfentanyl, and 4-fluoro-isobutyrylfentanyl. AAPS J. 2017;19(4):1102–22. https://doi.org/10.1208/s12248-017-0070-z.

    Article  CAS  PubMed  Google Scholar 

  6. Vikingsson S, Wohlfarth A, Andersson M, Gréen H, Roman M, Josefsson M, et al. Identifying metabolites of meclonazepam by high-resolution mass spectrometry using human liver microsomes, hepatocytes, a mouse model, and authentic urine samples. AAPS J. 2017;19(3):736–42. https://doi.org/10.1208/s12248-016-0040-x.

    Article  CAS  PubMed  Google Scholar 

  7. Diao X, Scheidweiler KB, Wohlfarth A, Pang S, Kronstrand R, Huestis MA. In vitro and in vivo human metabolism of synthetic cannabinoids FDU-PB-22 and FUB-PB-22. AAPS J. 2016;18(2):455–64. https://doi.org/10.1208/s12248-016-9867-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chimalakonda KC, Seely KA, Bratton SM, Brents LK, Moran CL, Endres GW, et al. Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands. Drug Metab Dispos. 2012;40(11):2174–84. https://doi.org/10.1124/dmd.112.047530.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hutter M, Moosmann B, Kneisel S, Auwarter V. Characteristics of the designer drug and synthetic cannabinoid receptor agonist AM-2201 regarding its chemistry and metabolism. J Mass Spectrom. 2013;48(7):885–94. https://doi.org/10.1002/jms.3229.

    Article  CAS  PubMed  Google Scholar 

  10. Asha S, Vidyavathi M. Cunninghamella—a microbial model for drug metabolism studies—a review. Biotechnol Adv. 2009;27(1):16–29. https://doi.org/10.1016/j.biotechadv.2008.07.005.

    Article  CAS  PubMed  Google Scholar 

  11. Diao X, Huestis MA. Approaches, challenges, and advances in metabolism of new synthetic cannabinoids and identification of optimal urinary marker metabolites. Clin Pharmacol Ther. 2017;101(2):239–53. https://doi.org/10.1002/cpt.534.

    Article  CAS  PubMed  Google Scholar 

  12. Guddat S, Fußhöller G, Beuck S, Thomas A, Geyer H, Rydevik A, et al. Synthesis, characterization, and detection of new oxandrolone metabolites as long-term markers in sports drug testing. Anal Bioanal Chem. 2013;405(25):8285–94. https://doi.org/10.1007/s00216-013-7218-1.

    Article  CAS  PubMed  Google Scholar 

  13. Tian J-L, Chen Y, Wang Y-X, Huang X-X, Sun X, Liu K-C, et al. Microbial transformation of methyl cyperenoate by Cunninghamella elegans AS 3.2028 and the antithrombotic activities of its metabolites. RSC Adv. 2016;6(113):112712–20. https://doi.org/10.1039/c6ra24332k.

    Article  CAS  Google Scholar 

  14. Watanabe S, Kuzhiumparambil U, Nguyen MA, Cameron J, Fu S. Metabolic profile of synthetic cannabinoids 5F-PB-22, PB-22, XLR-11 and UR-144 by Cunninghamella elegans. AAPS J. 2017;19(4):1148–62. https://doi.org/10.1208/s12248-017-0078-4.

    Article  CAS  PubMed  Google Scholar 

  15. Watanabe S, Kuzhiumparambil U, Winiarski Z, Fu S. Biotransformation of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella Elegans. Forensic Sci Int. 2016;261:33–42. https://doi.org/10.1016/j.forsciint.2015.12.023.

    Article  CAS  PubMed  Google Scholar 

  16. Nunomoto S, Kawakami Y, Yamashita Y, Takeuchi H, Eguchi S. Regioselectivity control in alkylation reactions of indolyl ambident anion. J Chem Soc Perkin Trans. 1990;(1, 1):111–4. https://doi.org/10.1039/P19900000111.

  17. Okauchi T, Itonaga M, Minami T, Owa T, Kitoh K, Yoshino H. A general method for acylation of indoles at the 3-position with acyl chlorides in the presence of dialkylaluminum chloride. Org Lett. 2000;2(10):1485–7. https://doi.org/10.1021/ol005841p.

    Article  CAS  PubMed  Google Scholar 

  18. Langer N, Lindigkeit R, Schiebel H-M, Ernst L, Beuerle T. Identification and quantification of synthetic cannabinoids in ‘spice-like’ herbal mixtures: a snapshot of the German situation in the autumn of 2012. Drug Test Anal. 2014;6(1–2):59–71. https://doi.org/10.1002/dta.1499.

    Article  CAS  PubMed  Google Scholar 

  19. Kavanagh P, Grigoryev A, Savchuk S, Mikhura I, Formanovsky A. UR-144 in products sold via the Internet: identification of related compounds and characterization of pyrolysis products. Drug Test Anal. 2013;5(8):683–92. https://doi.org/10.1002/dta.1456.

    Article  CAS  PubMed  Google Scholar 

  20. Shevyrin V, Melkozerov V, Nevero A, Eltsov O, Morzherin Y, Shafran Y. Identification and analytical properties of new synthetic cannabimimetics bearing 2,2,3,3-tetramethylcyclopropanecarbonyl moiety. Forensic Sci Int. 2013;226(1):62–73. https://doi.org/10.1016/j.forsciint.2012.12.009.

    Article  CAS  PubMed  Google Scholar 

  21. Sobolevsky T, Prasolov I, Rodchenkov G. Detection of urinary metabolites of AM-2201 and UR-144, two novel synthetic cannabinoids. Drug Test Anal. 2012;4(10):745–53. https://doi.org/10.1002/dta.1418.

    Article  CAS  PubMed  Google Scholar 

  22. Nielsen LM, Holm NB, Olsen L, Linnet K. Cytochrome P450-mediated metabolism of the synthetic cannabinoids UR-144 and XLR-11. Drug Test Anal. 2016;8(8):792–800. https://doi.org/10.1002/dta.1860.

    Article  CAS  PubMed  Google Scholar 

  23. Grigoryev A, Kavanagh P, Melnik A, Savchuk S, Simonov A. Gas and liquid chromatography-mass spectrometry detection of the urinary metabolites of UR-144 and its major pyrolysis product. J Anal Toxicol. 2013;37(5):265–76. https://doi.org/10.1093/jat/bkt028.

    CAS  PubMed  Google Scholar 

  24. Adamowicz P, Zuba D, Sekula K. Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine. Forensic Sci Int. 2013;233(1–3):320–7. https://doi.org/10.1016/j.forsciint.2013.10.005.

    Article  CAS  PubMed  Google Scholar 

  25. Cayman Chemical. 2017. https://www.caymanchem.com/Search?q=ur-144. Accessed 27 July 2017.

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Acknowledgments

The authors would like to thank Jane Cameron and Zofia Winiarski for the support with fungus culturing and Mahmoud El Safadi for the support with organic synthesis.

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Correspondence to Shanlin Fu.

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Watanabe, S., Kuzhiumparambil, U. & Fu, S. Structural Elucidation of Metabolites of Synthetic Cannabinoid UR-144 by Cunninghamella elegans Using Nuclear Magnetic Resonance (NMR) Spectroscopy. AAPS J 20, 42 (2018). https://doi.org/10.1208/s12248-018-0209-6

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