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Table of Contents
                            4-Methoxy- alpha -PVP: in silico prediction, metabolic stability, and metabolite identification by human hepatocyte incubation and high-resolution mass spectrometry
	Materials and methods
		Chemicals and reagents
		In silico metabolite prediction
		Metabolic stability of 4-methoxy- alpha -PVP in HLMs
		Incubation of 4-methoxy- alpha -PVP with cryopreserved primary human hepatocytes
		LC-HRMS for HLM samples
		LC-HRMS for human hepatocytes
		Data analysis
		In silico metabolite prediction
		Metabolic stability assessment with HLMs
		Identification of HLM metabolites
		Metabolite profiling with human hepatocytes
		Identification of O-demethylated metabolites
		Identification of hydroxylated metabolites
		Metabolite generated by ketone reduction
		Other identified metabolites
		Prevalence of 4-methoxy- alpha -PVP metabolites
		Metabolic stability assessment in HLMs
		Comparison with other synthetic cathinones
		In silico prediction of 4-methoxy- alpha -PVP metabolites
Document Text Contents
Page 1



4-Methoxy-a-PVP: in silico prediction, metabolic stability,
and metabolite identification by human hepatocyte incubation
and high-resolution mass spectrometry

Kayla N. Ellefsen1,2 • Ariane Wohlfarth1 • Madeleine J. Swortwood1 •

Xingxing Diao1 • Marta Concheiro1,3 • Marilyn A. Huestis1

Received: 10 June 2015 / Accepted: 12 July 2015 / Published online: 5 August 2015

� Japanese Association of Forensic Toxicology and Springer Japan (outside the USA) 2015

Abstract Novel psychoactive substances are continuously

developed to circumvent legislative and regulatory efforts. A

new synthetic cathinone, 4-methoxy-a-PVP, was identified for
the first time in illegal products; however, the metabolism of

this compound is not known. Complete metabolic profiles are

needed for these novel psychoactive substances to enable

identification of their intake and to link adverse effects to the

causative agent. This study assessed 4-methoxy-a-PVP
metabolic stability with human liver microsomes (HLMs) and

identified its metabolites after HLM and hepatocyte incuba-

tions followed by high-resolution mass spectrometry (HRMS).

A Thermo QExactive high-resolution mass spectrometer

(HRMS) was used with full scan data-dependent mass spec-

trometry, with (1) and without (2) an inclusion list of predicted

metabolite, and with full scan and all-ion fragmentation (3) to

identify potential unexpected metabolites. In silico predictions

were performed and compared to in vitro results. Scans were

thoroughly mined with different data processing algorithms

using WebMetabase (Molecular Discovery). 4-Methoxy-a-
PVP exhibited a long half-life of 79.7 min in HLM, with an

intrinsic clearance of 8.7 lL min-1 mg-1. In addition, this
compound is predicted to be a low-clearance drug with an

estimated human hepatic clearance of 8.2 mL min


Eleven 4-methoxy-a-PVP metabolites were identified, gener-
ated by O-demethylation, hydroxylation, oxidation, ketone

reduction, N-dealkylation, and glucuronidation. The most

dominant metabolite in HLM and human hepatocyte samples

was 4-hydroxy-a-PVP, also predicted as the #1 in silico
metabolite, and is suggested to be a suitable analytical target in

addition to the parent compound.

Keywords 4-Methoxy-a-PVP � Novel psychoactive
substances � Synthetic cathinones � Human hepatocytes �
Human liver microsomes � In silico prediction


In recent years, novel psychoactive substances (NPSs)

appeared rapidly on the drug market in an effort to bypass

controlled substance legislation. The European Monitoring

Centre for Drugs and Drug Addiction (EMCDDA) reported

41 NPSs identified for the first time across Europe in 2010,

81 in 2013, and 101 in 2014 [1]. These NPSs are contin-

uously developed to circumvent legislative and regulatory

efforts, with limited available pharmacological and toxi-

cological data. NPSs encompass a wide range of com-

pounds including synthetic cannabinoids, phenethylamines,

tryptamines, piperazines, ketamine, cathinones, and other

plant-based psychoactive substances [2].

Synthetic cathinones emerged on the designer drug

market as popular ‘‘legal’’ alternatives to illicit drugs in the

late 2000s, and are marketed as ‘‘legal highs’’ and ‘‘not for

human consumption’’. They are stimulant-like drugs

derived from cathinone, the active ingredient of the khat

plant Catha edulis, with adverse effects including hyper-

thermia, agitation, confusion, psychosis, seizures, and

& Marilyn A. Huestis
[email protected]

Chemistry and Drug Metabolism, Intramural Research

Program, National Institute on Drug Abuse, National

Institutes of Health, 251 Bayview Boulevard, Suite 200

Room 05A-721, Baltimore, MD 21224, USA

Program in Toxicology, University of Maryland Baltimore,

Baltimore, MD, USA

Department of Sciences, John Jay College of Criminal

Justice, City University of New York, New York, NY, USA


Forensic Toxicol (2016) 34:61–75

DOI 10.1007/s11419-015-0287-4

Page 2

tachycardia [3–7]. A variety of synthetic cathinones, alone

and in combination with other illicit drugs, were detected

in acute intoxications [4, 8, 9], impaired driving cases [10–

13], and fatalities [4, 12, 14–23].

4-Methoxy-a-PVP (4-methoxy-a-pyrrolidinovalerophe-
none) was identified for the first time in illegal products

purchased in Japan in 2013 [24] and also recently identified

by Customs in Berlin [25]. 4-Methoxy-a-PVP is a substi-
tuted cathinone containing a pyrrolidinyl moiety and a

methoxy group on the 40 position of the aromatic ring
(Fig. 1). Its structure is similar to other a-pyrrolidinophe-
none derivatives such as 3,4-methylenedioxypyrovalerone

(MDPV) and a-pyrrolidinovalerophenone (a-PVP), both
schedule I compounds [26, 27]. The mechanism of action

of 4-methoxy-a-PVP is unknown; however, based on
structural similarities to MDPV and a-PVP, it is hypothe-
sized that 4-methoxy-a-PVP would act similarly as a
monoamine transporter blocker increasing the amount of

extracellular dopamine [28–31]. Extracellular dopamine

increases pose a higher risk for addiction [28, 31]. In

addition, the high lipophilicity of the pyrrolidine ring

increases permeability through the blood–brain barrier, and

hence increased potency and abuse potential [29, 32, 33].

The metabolic pathways of other a-pyrrolidinophenones,
including MDPV, a-PVP, a-pyrrolidinobutiophenone

(a-PBP), a-pyrrolidinopropiophenone (a-PPP), 4-methyl-
a-pyrrolidinopropiophenone (MPPP), and 4-methoxy-a-
pyrrolidinopropiophenone (MOPPP), were previously

investigated [34–42]. There are no metabolism studies for

4-methoxy-a-PVP. A recent review of the pharmacology of
a-pyrrolidinophenones outlined the major metabolic path-
ways of other structurally similar compounds including

reduction of the keto moiety to the corresponding alcohol,

hydroxylation followed by oxidation of the pyrrolidine ring

to the lactam (200-oxo), hydroxylation and carboxylation of
the 40-methyl group, O-demethylation of the 40-methoxy
group, and demethylenation followed by O-methylation of

the 30, 40-methylenedioxy moiety [43].
A promising approach to elucidate metabolites of NPSs

includes in silico metabolite predictions, and human liver

microsome (HLM) and human hepatocyte incubations,

followed by analysis with high-resolution mass spec-

trometry (HRMS) and software-assisted data mining [44,

45]. Complete metabolic profiles are needed for 4-meth-

oxy-a-PVP to enable identification of intake and link
adverse effects to the causative agent. We evaluated

4-methoxy-a-PVP in silico metabolism predictions,
assessed metabolic stability with HLMs, and identified

metabolites after HLM and human hepatocyte incubations

followed by HRMS.

Fig. 1 Product ion mass spectrum of 4-methoxy-a-PVP and its fragmentation pattern

62 Forensic Toxicol (2016) 34:61–75


Page 7

Fig. 2 Product ion mass spectra
and assigned fragmentation

patterns for 4-methoxy-a-PVP
metabolites generated by O-

demethylation. a 4-Hydroxy-a-
PVP M3, b O-demethylated and
ketone reduced metabolite M2,

and c the O-demethylated and
N-dealkylated metabolite M1

Forensic Toxicol (2016) 34:61–75 67


Page 8

Fig. 3 Product ion mass spectra
and assigned fragmentation

pattern for hydroxylated

4-methoxy-a-PVP for
metabolites: a the di-
hydroxylated metabolite on the

pyrrolidine ring M6, b the
monohydroxylated metabolite

on the pyrrolidine ring M4, and

c the ring opening and
hydroxylated metabolite M5.

The exact location of the di-

hydroxylated metabolite is

unknown; however, the position

of the hydroxyl group at the 200

in M4 is suggested based on

previously identified a-
pyrrolidine metabolites

68 Forensic Toxicol (2016) 34:61–75


Page 14

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