ISSUE 2 Cayman NPS Monograph Nathan K. Layle, Ting Zhao, and Donna M. Iula

N-butyl Pentylone Item No. 26701 Synthetic Cathinone

O H O H HO N N O HO M1 A Amine group O Aryl ring O O H NH O N B O 2

N-butyl Pentylone is a synthetic cathinone O O first reported in 1960s German patent literature, M2 emerging on the recreational market Ketone C O in late 2018.1 Cathinone analogs have seen H O N widespread abuse since the mid-2000s and (A) Demethylenation O are derived from the alkaloid cathinone found OH 2 (B) N-Dealkylation M3 in khat. N-butyl Pentylone has been identified (C) on illicit research chemical websites and in Observed phase I metabolites of N-butyl pentylone seized drug samples. While several similar detected using high mass accuracy fragments cathinone analogs are Schedule I controlled generated by LC-MS/MS. substances, N-butyl pentylone remains uncontrolled as of mid-2020. The goal of this monograph is to identify its probable phase I metabolites using HLM assays. This study can provide insights into the metabolic pathway of N-butyl pentylone, and by analysis with Need a Reference Standard for a high-resolution Orbitrap mass spectrometry, we Specific Metabolite? aim to provide forensic toxicologists with the key ions/fragments that can help identify this Have a Request for Future Studies? novel synthetic cathinone in biological samples. Contact Us at [email protected]

Cayman Chemical · (800) 364-9897 www.caymanchem.com 1180 E. Ellsworth Road · Ann Arbor, MI · 48108 Mass Spectrum and Fragments of N-butyl Pentylone

O O O O H H H H2 N N O N N O O O O O O O Exact Mass: 128.1434 Exact Mass: 149.0233 Exact Mass: 205.0859 Exact Mass: 230.1539 Exact Mass: 260.1645 Exact Mass: 278.1751

O O O O H H H H2 N N O N N O O O O O O O Exact Mass: 128.1434 Exact Mass: 149.0233 Exact Mass: 205.0859 Exact Mass: 230.1539 Exact Mass: 260.1645 Exact Mass: 278.1751

O O O O H H H H2 N N O N N O O O O O O O Exact Mass: 128.1434 Exact Mass: 149.0233 Exact Mass: 205.0859 Exact Mass: 230.1539 Exact Mass: 260.1645 Exact Mass: 278.1751

O O O O H H H H2 N N O N N O O O O O O O O O O H H O H H 2 N N O N N O O O Exact Mass:O 128.1434 Exact Mass: 149.0233 Exact Mass: 205.0859 Exact Mass: 230.1539 Exact Mass: 260.1645 Exact Mass: 278.1751 O O O Exact Mass: 128.1434 Exact Mass: 149.0233 Exact Mass: 205.0859 Exact Mass: 230.1539 Exact Mass: 260.1645 Exact Mass: 278.1751

Figure 1. High-resolution mass spectrum of the parent compound N-butyl pentylone with proposed fragment structures.

N-butyl Pentylone Metabolite Formation and Detection The parent compound N-butyl pentylone was incubated with human microsomes (HLM) to form phase I metabolites. An aliquot from the incubation was quenched with acetonitrile, and the mixture was centrifuged at 13,200 rpm for 10 minutes. The obtained supernatant was injected onto a Dionex Ultra-High Performance Liquid Chromatography (UHPLC) system for metabolite separation. The eluted metabolites were detected using a high-resolution Orbitrap mass spectrometer (Thermo Scientific). For comparison, a control sample was prepared by incubating N-butyl pentylone with deactivated HLM. The total ion chromatogram () along with extracted ion chromatograms (EICs) of the incubation compared with those of the control were used to identify metabolites formed from the HLM assay with N-butyl pentylone. The TIC shows potential metabolites at 4.75 (M1), 4.80 (M2), and 5.09 (M3) min retention times (RT), as well as the parent, N-butyl pentylone, at 5.70 min. The EICs show the three main phase I metabolites by their exact masses, formed from our HLM incubation. All three metabolites gave masses consistent with the known ways 3,4-methylenedioxycathinones are metabolized: demethylenation, N-dealkylation, and oxidation (hydroxylation).3 In our study, we did not observe the possible hydrogenation metabolite that results from reduction of the β-ketone to the corresponding that can occur with some cathinones. This result agrees with the literature on the metabolism of 3,4-methylenedioxy-substituted cathinones.3 Interestingly, we observed two peaks at 5.38 min and 5.70 min in the metabolite incubation that correspond with the MS/MS spectrum of the parent compound. However, we only observed a single peak of the parent compound at 5.72 min in the control sample. The earlier 5.38 min peak displays no evidence of a mass shift, indicating that it results from a change in the parent N-butyl pentylone because of incubation and not as the result of metabolism.

2 TIC

M1 & M2

N-butyl Pentylone 5.70 min

M3

4.75 4.80 M1 EIC M2 EIC 266.1751 m/z 222.1125 m/z

5.09 M3 EIC 294.1700 m/z

5.00 5.21

Figure 2. TIC (top) and EICs (bottom) of N-butyl pentylone post-incubation. EICs and corresponding exact masses for metabolites M1, M2, and M3 represent demethylenation, N-dealkylation, and hydroxylation, respectively.

3 Metabolite 1 (M1) The first eluting metabolite (4.75 min) yielded the MS/MS spectrum below with proposed major fragments. This demethylenation metabolite is consistent with literature metabolism data on structurally similar analog N-ethyl pentylone, which includes authentic human biological samples.4 Two key ions generated from fragmentation of this metabolite are 266.1743 m/z and 193.0857 m/z, both of which show the

loss of the methylenedioxy methylene (CH2) group. O O H HO N O O H H2 H2 N N HO N HO O HO H3N + + : C11H13O3 Chemical Formula: C15H20NO Exact Mass: 74.0964 Exact Mass: 128.1434 Exact Mass: 193.0859 Exact Mass: 230.1539 Exact Mass: 248.1645 Exact Mass: 266.1751

O O H HO N O O H H2 H2 N N HO N HO O HO H3N + + Chemical Formula: C11H13O3 Chemical Formula: C15H20NO Exact Mass: 74.0964 Exact Mass: 128.1434 Exact Mass: 193.0859 Exact Mass: 230.1539 Exact Mass: 248.1645 Exact Mass: 266.1751

O O H HO N O O H H2 H2 N N HO N HO O HO H3N O O O + O + H Chemical Formula: C11H13O3 Chemical Formula:H C15H20NO HO N HO N Exact Mass: 74.0964 Exact Mass: 128.1434 Exact Mass: 193.0859 O Exact Mass: 230.1539 O Exact Mass:O 248.1645 Exact Mass:O 266.1751 H H H2 H2 H2 H2 N N O O N HO N N HO N HO HO H HO O N O O O H HO H2 H2 HO H3N N + + N HO N ChemicalH3N Formula: C H O Chemical Formula: C H NO + + 11 13 3 HO Chemical15 20 Formula: C11H13O3 Chemical Formula: C15H20NO O Exact Mass: 74.0964 Exact Mass: 128.1434 ExactExact Mass: Mass: 74.0964 193.0859 Exact Mass:Exact 128.1434 Mass: 230.1539Exact Mass: 193.0859Exact Mass: 248.1645 Exact Mass: 230.1539Exact Mass: 266.1751Exact Mass: 248.1645 Exact Mass: 266.1751 HO H3N + + Chemical Formula: C11H13O3 Chemical Formula: C15H20NO Exact Mass: 74.0964 Exact Mass: 128.1434 Exact Mass: 193.0859 Exact Mass: 230.1539 Exact Mass: 248.1645 Exact Mass: 266.1751

Figure 3. High-resolution MS/MS spectrum of demethylenation metabolite M1 and proposed fragment structures.

4 Metabolite 2 (M2) The second eluting metabolite had a retention time of 4.80 min and yielded the MS/MS spectrum and fragments below. The key fragments that indicate N-dealkylation include 222.1120 m/z, 204.1016 m/z, and 174.0912 m/z. These fragments have proposed structures based on the fragmentation of similar cathinones.

O O O NH2 NH2 O NH3 O NH2 O O H2N O Exact Mass: 72.0808 Exact Mass: 135.0441 Exact Mass: 146.0964 Exact Mass: 174.0913 Exact Mass: 204.1019 Exact Mass: 222.1125

O O O NH2 NH2 O NH3 O NH2 O O H2N O Exact Mass: 72.0808 Exact Mass: 135.0441 Exact Mass: 146.0964 Exact Mass: 174.0913 Exact Mass: 204.1019 Exact Mass: 222.1125

O O O NH2 ONH2 O NH3O O O O O O NH2 NH2 NH2 O NH3 O NH O O NH O NH O NH2 O 2 2 O 3 H N 2 O O NH2 NH2 O NH2 O O NH3 H N O Exact Mass: 72.0808 Exact Mass: 135.04412 Exact Mass:O 146.0964O Exact Mass: 174.0913NH2 Exact Mass: 204.1019 Exact Mass: 222.1O 125 H2N O Exact Mass: 72.0808 Exact Mass: 135.0441 Exact Mass: 146.0964 Exact Mass: 174.0913 O Exact Mass: 204.1019 O Exact Mass: 222.1125 H2N Exact Mass: 72.0808 O Exact Mass: 135.0441 Exact Mass: 146.0964 Exact Mass: 174.0913 Exact Mass: 204.1019 Exact Mass: 222.1125 Exact Mass: 72.0808 Exact Mass: 135.0441 Exact Mass: 146.0964 Exact Mass: 174.0913 Exact Mass: 204.1019 Exact Mass: 222.1125

Figure 4. High-resolution MS/MS spectrum of N-dealkylation metabolite M2 and proposed fragment structures.

5 Metabolite 3 (M3) The latest eluting metabolites had retention times of 5.00 min/5.09 min/5.21 min and yielded the averaged mass spectrum and proposed fragments below. This spectrum represents the various possible mono-hydroxylation metabolites. All three metabolites yielded the two key fragments with 294.1693 m/z and 276.1587 m/z, representing the hydroxylation product and elimination fragment. However, each peak showed different abundances of the ions 258.1482 m/z, 234.1119 m/z, and 216.1014 m/z, indicating possible differences in the position of hydroxylation. As reported in the literature, the hydroxylation of 3,4-methylenedioxy-substituted cathinones occurs predominately on the ketone acyl chain and not on the N-alkyl group.5

O O O O H2 H2 O N H O N O O O O N O O OH O + O Chemical Formula: C12H13O3 Exact Mass: 135.0441 Exact Mass: 205.0859 Exact Mass: 234.1125O Exact Mass: 276.1594 Exact Mass: 294.1700 O O O H2 H2 O N H O N O O O O N O O OH O + O Chemical Formula: C12H13O3 Exact Mass: 135.0441 Exact Mass: 205.0859 Exact Mass: 234.1125 Exact Mass: 276.1594 Exact Mass: 294.1700

O O O O H2 H2 O N H O N O O O O N O O OH O + O Chemical Formula: C12H13O3 Exact Mass: 135.0441 Exact Mass: 205.0859 Exact Mass: 234.1125 Exact Mass: 276.1594 Exact Mass: 294.1700

O O O O O H2 H2 O N H O O N O O O H2 H2 O N O O O O N O N H O O O O O N OH O Chemical Formula: C H O + O O O 12 13 3 OH O Exact Mass: 135.0441 Exact+ Mass:O 205.0859 Exact Mass: 234.1125 Exact Mass: 276.1594 Exact Mass: 294.1700 Chemical Formula: C12H13O3 Exact Mass: 135.0441 Exact Mass: 205.0859 Exact Mass: 234.1125 Exact Mass: 276.1594 Exact Mass: 294.1700

Figure 5. High-resolution MS/MS spectrum of hydroxylation metabolites M3 and proposed fragment structures.

6 Conclusion Incubation of N-butyl pentylone with HLM resulted in three different phase I metabolites (M1, M2, M3). Based on MS/MS fragmentation data, we can conclude that the first of the observed metabolites is the result of demethylenation of the 3,4-methylenedioxy moiety. The second observed metabolite occurs as the result of N-dealkylation of the butylamine group. The third set of metabolites arises from hydroxylation at possible sites along the pentanone chain. These metabolites can all serve as biomarkers for cathinone abuse, and our results indicate that the N-dealkylation and demethylenation metabolites are the most abundant in our HLM study. The parent compound is also a valuable indicator in the case of 3,4-methylenedioxy-substituted cathinones because of their greater stability.6

Methodology and Instrumentation In vitro metabolism studies The compound N-butyl pentylone was diluted to the necessary concentration by preparing a 50 mM stock DMSO solution and further diluting with phosphate buffer (pH 7.4). The compound solution was then incubated for two hours at 37ºC with HLM and the added cofactor NADPH. At two hours, the reaction was quenched with acetonitrile and spun down on a centrifuge. An aliquot of the mixture was injected onto the UHPLC. The eluates were detected using an Orbitrap mass spectrometer. · Microsomes: 50-pool mixed-gender microsomes, 20 mg/ml protein conc. (Sekisui XenoTech, H0610) · Analyte: N-butyl pentylone (hydrochloride) (Cayman Chemical, Item No. 26701) · Incubation time of two hours · Sample prepared in pH 7.4 phosphate buffer, 50 mM stock DMSO solution · Instrumentation and analysis (LC/MS-MS): – Dionex™ UltiMate™ 3000 UHPLC (Thermo Scientific™) – Acquity UPLC® BEH C8 column, 1.7 μm, length 2.1 x 100 mm (Waters™) – Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific™) – Xcalibur™ software v. 4.0 (Thermo Scientific™)

References 1. Koeppe, H., Ludwig, G., and Zeile, K. Substituted phenyl-α-amino ketones. Boehringer, C. H., Sohn. DE1242241 (1967). 2. Majchrzak, M., Celiński, R., Kuś, P., et al. The newest cathinone derivatives as designer : An analytical and toxicological review. Forensic Toxicol. 36(1), 33-50 (2018). 3. Uralets, V., Rana, S., Morgan, S., et al. Testing for designer stimulants: Metabolic profiles of 16 synthetic cathinones excreted free in human urine. J. Anal. Toxicol. 38(5), 233-241 (2014). 4. Krotulski, A.J., Papsun, D.M., De Martinis, B.S., et al. N-ethyl pentylone (ephylone) intoxications: Quantitative confirmation and metabolite identification in authentic human biological specimens. J. Anal. Toxicol. 42(7), 467-475 (2018). 5. Mueller, D.M. and Rentsch, K.M. Generation of metabolites by an automated online metabolism method using human liver microsomes with subsequent identification by LC-MS(n), and metabolism of 11 cathinones. Anal. Bioanal. Chem. 402(6), 2141-2151 (2012). 6. Kerrigan, S. and Glicksberg, L. Long-term stability of synthetic cathinones in forensic toxicology samples. National Criminal Justice Reference Service (2017).

7 WALL POSTERS Cayman's scientists have developed these laboratory guides for toxicologists and drug chemists to help identify clinically relevant metabolites from several major classes of and . Request free copies for your lab at www.caymanchem.com/posters.

SYNTHETIC CANNABINOID METABOLISM LAB GUIDE METABOLISM LAB GUIDE

Amide Linker/Fluoropentyl Tail Linker/Pentyl Tail Amide Linker/Fluorobenzyl Tail Amide Linker/Cyclohexylmethyl Tail 7-Halogenated-1,4-Benzodiazepines 7-Nitro-1,4-Benzodiazepines Half-Life of Common Benzodiazepines

HO NH OH NH2 NH2 OH OH OH 2 O H H + + + NH2 NH O H O O O Drug (Brand Name) Half-Life (h) Half-LifePrimary (hr) Metabolite (half-life, h) 2 N N N N N O O O O O O O O O O O O O O NH O O NH NH NH NH NH NH O O O O Long Half-Life (effective >24 h)hr) NH NH OH OH OH N N N C N A N N C N A R N N N N N (Valium) 20-100 20-100Nordiazepam (36-200) N N B O2N H2N N N N A N N N N N N A NH2 N N H H OH R' R R R R (Librium) (Librium) 5-30 5-30Nordiazepam (36-200) O O F OH F F F CE B NH C H AB-CHMINACA (Tranxene) * * Nordiazepam (36-200) AB-PINACA AB-FUBINACA 1 O O HO N N N NH2 Biotransformation Pathway Biotransformation Pathway NH O 2 (Centrax) * * Nordiazepam (36-200) N OH OH OH A O O C C D NH2 NH2 NH O O H NH 7 N 4 N (Dalmane) * * Desalkylflurazepam (40-250) F B N R R H H O O O O B NH NH O O N N NH NH N N R' R' Intermediate to Short Half-Life (effective 5-24 h)hr) E A N 5-fluoro AB-PINACA N N H H N N H Drug O 1 O O O Drug N N F O (Alodorm) 15-38 15-387-Aminonitrazepam N N N N N N N OMe OH OH OH OH (Rohypnol) 20-30 20-307-Aminoflunitrazepam O AKB48 7 4 O O B N O2N N O2N N H2N N H2N N NH O O R (Restoril) 8-22 8-22Oxazepam (4-15) OH NH A OMe OH OH R OMe OH R R R R (Lectopam) 10-20 10-203-hydroxy Bromazepam O O N N R' A CE B O O O O O O O O NH C O O O O NH NH NH NH N N NH NH (Ativan) 10-20 10-20Lorazepam Glucuronide N N C N B N N C F F N C N Drug Primary Metabolite H CH CH F CO R CONH NCH R N H CH3 CO2H CO2R NO2 3 2 2 2 2 N N N N N N (Xanax) 6-20 6-20α-hydroxy Alprazolam MMB-FUBINACA (AMB-FUBINACA · FUB-AMB) Examples CE F F OH Amide-linked head groups with the t-butyl group are B (Serax) 4-15 4-15Oxazepam Glucuronide R=NH2, AB-PINACA O O O H O (Etizest) 3.4-7.1 3.4-7.1α-hydroxy Etizolam A A B C C D 5-fluoro AMB R=OMe, AMB (MMB-PINACA) metabolized similarly to MMB-FUBINACA. MA-CHMINACA A B C D E N N N N Examples H Ultra-Short Half-Life (effective <5 h)hr) Cl N Cl N Cl N Br N O H O H O O O N N N N N (Versed) <5 <5α-hydroxy Midazolam Cl F Cl

OH O2N N O2N N O2N N (Halcion) <5 <5α-hydroxy Triazolam N N Phase I Metabolite Phase I Metabolite Diazepam* H2N O2N F Cl

+ Ester Linker/Fluoropentyl Tail Ester Linker/Pentyl Tail Ester Linker/Fluorobenzyl Tail Ester Linker/Cyclohexylmethyl Tail H O H O H O O R R Approximate half-life data adapted from Greenblatt, D.J., Shader, R.I., Divoll, M., et al. Br. J. Clin. Pharmacol. N N N N Flunitrazepam Nitrazepam 11 (Suppl 1), 11S-16S (1981) and Manchester, K.R., Lomas, E.C., Waters, L., et al. Drug Test Anal. 10(1), 37-53 (2018). OH OH OH O O O *Indicates or drug precursors that do not reach the systemic circulation in clinically important amounts. OH OH OH Cl N Cl N Cl N Cl N O O N O O O OH Cl O OH A N C N N OH N C O N Nordiazepam Lorazepam Oxazepam Temazepam F N N N O C C N N OH H H CO H NH OH CH OH CH OH CO H CO H NH O N 2 2 2 2 2 2 O F F APINAC N *The primary metabolite of diazepam is nordiazepam Benzodiazepine Prodrugs FUB-PB-22 N A via phase I demethylation. A O N B C O N N N H O H O H O F BB-22 F G H HO N N N E E O N N N N O CO2Et CO2H H 5-fluoro PB-22 O N N N O N H H H O OH A A OH Cl N D Cl N CE Cl N OH N R N R N R N N F F F OH N Phase II Metabolite R' R' R' N F All ester-linked synthetic cannabinoids predominantly OH FDU-PB-22 Loflazepate Desalkylflurazepam undergo hydrolysis to cleave the head group. N N N HO N N N S N S N HO S N H H O O H O Examples N N CO2H N N N N O NH CO2H H N N N N OH H H Carbonyl Linker/Fluoropentyl Tail Carbonyl Linker/Pentyl Tail Typical Metabolism Patterns OH H3C R A R A R N N N N O O N N N N Cl N Cl N Cl N

Cl N Cl N O N N Br N · All regions (head/core/tail) of classic synthetic cannabinoids can undergo metabolism. HO2C OH 2 CE A Hydroxylation Cl Cl F A D Dealkylation O O O O · All hydroxylated metabolites can be further oxidized. OH (MW plus 16) Clorazepate Nordiazepam Oxazepam E E - Internal secondary can oxidize to their corresponding ketones. Examples Alprazolam Triazolam Hydrolytic Dehalogenation N N N N - Terminal hydroxy metabolites can oxidize to the carboxylic acid. B E Oxidation B A (MW minus 2) OH OH OH OH N N N O O O O · The nature of amide-linked head groups (terminal amide vs. terminal ester) dictates metabolic fate. O N N N 8-Nitrotriazolobenzodiazepines can also undergo Hydroxylation Reduction S N S N S N S N · All indole/indazole cores can be hydroxylated, although these are minor products. A E Cl Br reduction to 8-aminotriazolobenzodiazepines. O (MW plus 16) (MW minus 30) N N N N N C Hydrolysis N N A O O A O O · All synthetic cannabinoids containing N-alkyl chains can undergo hydroxylation on the chain. Dealkylation What is a ? Demethylation Sulfation Cl Cl F Cl F & B F Cl N A prodrug is a drug substance that needs to be E · The 4-fluorobenzyl tail does not undergo metabolism. (MW minus 14) (MW plus 80) AM2201 HO N N OH JWH 018 N OH N O Etizolam Fluclotizolam converted into the pharmacologically active agent · 5-Fluoropentyl tails can be hydroxylated at the ω-1 site. They can also undergo oxidative defluorination 1 F F Decarboxylation View a complete list of parent compounds, metabolites, by metabolic or physiochemical transformation. to form the same 5-hydroxypentyl metabolite as N-pentyl tail synthetic cannabinoids. C G Thienodiazepines contain a ring in place of the View primary resources used to collate this guide 1. Drago, D., Pacifici, E., and Bain, S. Chapter 3 Drugs. An overview of FDA regulated products: From Drugs (MW minus 44) (MW plus 176) ring of a standard benzodiazepine but exhibit and labeled standards at www.caymanchem.com Prazepam at www.caymanchem.com/SCmetabolism · All initial metabolites can undergo further biotransformations. and cosmetics to food and tobacco. Pacifici, E. and Bain, S., editors, 1st edition, Academic Press (2018). Acetylation similar activity and pharmacological effects. D Hydrolysis H © Cayman Chemical Company · Created April 2020 (MW plus 42) © Cayman Chemical Company · Created June 2019

Synthetic Cannabinoid Benzodiazepine Metabolism Lab Guide Metabolism Lab Guide

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