Evidence of enzyme-mediated transesterification of synthetic cannabinoids with ethanol: potential toxicological impact

Article abstract of DOI:10.1007/s11419-019-00491-0

Purpose: Synthetic cannabinoids (SCs) represent a large proportion of novel psychoactive substances on the black market and have caused a number of deaths. Polydrug use including combination of SCs and ethanol could further complicate the toxicological impact. To the best of our knowledge, there have been no reports presenting evidence of transesterification between SCs and ethanol in vitro. Methods: The in vitro metabolism of the four carboxylate SCs PB-22, NPB-22, 5-fluoro-PB-22 (5F-PB-22), and 5-fluoro-NPB-22 (5F-NPB-22) in the presence of ethanol using human liver microsomes with and without appropriate enzyme inhibitors was studied. Newly identified SC ethyl esters were chemically synthesised and fully characterised. The activity of these SCs and their ethanol transesterification products were assessed using cannabinoid receptor (CB₁ and CB₂) activation assays. Results: SCs/ethanol transesterification products were detected and studied using liquid chromatography–high-resolution mass spectrometry. We have shown that the SC ethyl ester formation is mediated by human carboxyl esterase enzymes. The ethyl esters exhibited a reduced activity for the CB receptors compared with their parent compounds. Conclusions: These novel ethyl esters may be useful additional markers of cannabinoid administration, and especially so if they prove to have longer half-lives than their parent compounds.

Full text of DOI:10.1007/s11419-019-00491-0

Forensic Toxicology
https://doi.org/10.1007/s11419-019-00491-0
ORIGINAL ARTICLE
Evidence of enzyme‑mediated transesteriꢀcation of synthetic
cannabinoids with ethanol: potential toxicological impact
1
1,2
1,3
1,2
4
Orapan Apirakkan · Ivana Gavrilović · Giuseppe Floresta · Cheyanne Pierre · Annelies Cannaert ·
4
5,6
1
7
1
Christophe P. Stove · Paul I. Dargan · David A. Cowan · Lewis Couchman · Vincenzo Abbate
Received: 21 May 2019 / Accepted: 2 July 2019
©
The Author(s) 2019
Abstract
Purpose Synthetic cannabinoids (SCs) represent a large proportion of novel psychoactive substances on the black market
and have caused a number of deaths. Polydrug use including combination of SCs and ethanol could further complicate the
toxicological impact. To the best of our knowledge, there have been no reports presenting evidence of transesteriꢀcation
between SCs and ethanol in vitro.
Methods The in vitro metabolism of the four carboxylate SCs PB-22, NPB-22, 5-ꢁuoro-PB-22 (5F-PB-22), and 5-ꢁuoro-
NPB-22 (5F-NPB-22) in the presence of ethanol using human liver microsomes with and without appropriate enzyme inhibi-
tors was studied. Newly identiꢀed SC ethyl esters were chemically synthesised and fully characterised. The activity of these
SCs and their ethanol transesteriꢀcation products were assessed using cannabinoid receptor (CB and CB ) activation assays.
1
2
Results SCs/ethanol transesteriꢀcation products were detected and studied using liquid chromatography–high-resolution
mass spectrometry. We have shown that the SC ethyl ester formation is mediated by human carboxyl esterase enzymes. The
ethyl esters exhibited a reduced activity for the CB receptors compared with their parent compounds.
Conclusions These novel ethyl esters may be useful additional markers of cannabinoid administration, and especially so if
they prove to have longer half-lives than their parent compounds.
Keywords Carboxylate synthetic cannabinoids · In vitro drug metabolism · Transesteriꢀcation · 5F-NPB-22 · Ethanol ·
Biomarkers
Introduction
2008–2015 [1] under the pretence that these compounds are
safe’ and ‘legal’ [2], and it is assumed that their use cannot
‘
The use of synthetic cannabinoid receptor agonists, or sim-
ply ‘synthetic cannabinoids’ (SCs), is a global toxicologi-
cal conundrum. Their use appears to have escalated during
easily be detected [3]. Although there has been a decline
in seized SCs recently, many of the recent substances are
highly potent and harmful [1]. Originally, these compounds,
which circumvented drug legislation in many countries,
were sold over-the-counter in so-called headshops and on
the Internet as vividly branded products such as ‘Spice’ in
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11419-019-00491-0) contains
supplementary material, which is available to authorized users.
4
*
Vincenzo Abbate
Laboratory of Toxicology, Department of Bioanalysis,
vincenzo.abbate@kcl.ac.uk
Faculty of Pharmaceutical Sciences, Ghent University,
Ottergemsesteenweg 460, 9000 Ghent, Belgium
1
Department of Analytical, Environmental and Forensic
Sciences, King’s Forensics, King’s College London,
London SE1 9NH, UK
5
Clinical Toxicology, Guy’s and St Thomas’ NHS Foundation
Trust and King’s Health Partners, London, UK
6
Clinical Toxicology, Faculty of Life Sciences and Medicine,
2
3
Department of Analytical, Environmental and Forensic
Sciences, Drug Control Centre, King’s Forensics, King’s
College London, London SE1 9NH, UK
King’s College London, London, UK
7
Analytical Services International, St. George’s University
of London, Cranmer Terrace, London SW17 0RE, UK
Department of Drug Sciences, University of Catania, V.le
A. Doria, 95125 Catania, Italy
Vol.:(0
1
123456789)
3

Forensic Toxicology
Europe and ‘K2’ in the United States. The weakness of the
Misuse of Drugs Act [4] and incorporated schedules in the
UK could not readily keep up with the rapidity of novel
compounds reaching the market, but the more recent UK
Psychoactive Substances Act (PSA) in 2016 [5] appears to
be having an impact. While some data suggest that the use
of novel psychoactive substances (NPS), including SCs, has
decreased since the introduction of the PSA, other studies
suggest little impact in the number of emergency department
presentations, and their use remains prevalent and problem-
atic [6]. As of the end of 2017, more than 670 NPS were
monitored by the European Monitoring Centre for Drugs
and Drug Addiction (EMCDDA), and 179 out of these were
SCs [1]. In the UK, this is especially relevant in the prisoner
population and amongst those facing homelessness [3, 7].
Not only are the clinical features of acute SC toxicity dif-
(GC–MS) in the presence of methanol or ethanol as a sol-
vent [28].
There is increasing evidence conꢀrming the use of SCs
together with ethanol. A recent United Nations Oꢂce on
Drugs and Crime (UNODC) report [29] highlighted ethanol
as the third most frequent substance found in SC-related
fatalities. In addition, ester-based SCs including 5F-PB-22
were detected, together with ethanol and other substances in
postmortem cases [30, 31]. ADB-CHMINACA (also known
as MAB-CHMINACA) and ethanol were both detected in
a patient admitted to hospit [32]. A further case reported a
fatal poisoning with the synthetic cannabinoid AB-CHMI-
NACA and ethanol [33]. Finally, blood and urine ethanol
concentrations were reported in three fatalities related to
the use of 5F-ADB, 5F-PB-22 and AB-CHMINACA (5F-
PB-22 = 2.60 and 3.58 g/kg, AB-CHMINACA = 1.45 and
2.57 g/kg, 5F-ADB=0.09 and 0.12 g/kg for blood and urine
ethanol concentrations, respectively) [34]. It is apparent that
the discovery of novel biomarkers of concomitant SC-etha-
nol consumption should facilitate the interpretation of such
analytical investigations and may allow both extending the
detection time and assisting in the understanding of the pat-
terns of toxicity associated with such polydrug abuse.
In this study, we report in vitro evidence from HLM incu-
bation experiments for the transesteriꢀcation of four ester-
containing SCs, namely PB-22, NPB-22, 5F-PB-22, and
5F-NPB-22 (Fig. 1), in the presence of ethanol, giving rise
to a further group of potential new indicators of combined
SC/ethanol use. These compounds were chosen because of
the presence of a labile ester bond in their structures, making
them amenable to the described chemical reaction, as well
as their prevalence [35].
ꢀ
cult to identify and treat, particularly for the more recent
third-generation SCs [6, 8, 9], but the analysis of these com-
pounds in bulk-drug seizures and in biological samples can
also be challenging, even using the most modern analytical
instrumentation. Analysis is complicated by (1) the sheer
number of SCs that exist [10, 11], (2) their physicochemical
diversity, (3) the frequent formation of multiple (pharmaco-
logically active) metabolites [10, 12–14], (4) their (in)stabil-
ity and chemical transformations when ingested via smoking
(
i.e., their pyrolysis products [15–18]), (5) their potency,
which transpires to low concentrations in biological samples,
and (6) the limited availability of reference materials. To
aid the identiꢀcation of SCs and their metabolites, in vitro
models based on incubation with human liver microsomes
(
HLMs) and other enzyme fractions [19], coupled with in
silico predictions, are commonly used to determine the most
likely metabolic routes of ‘new’ SCs [20]. In silico models
are also extremely useful at predicting the pharmacology
and potential toxicology of SCs [21, 22]. Such literature
resources are invaluable for building large screening librar-
ies to maximise the chances of detecting toxicologically
relevant SC use in samples.
To evaluate whether the transesteriꢀcation products might
be more biologically active than the parent compounds, we
used an activity-based cannabinoid receptor assay [36–38]
to investigate the in vitro receptor activity of the ethyl esters
formed by transesteriꢀcation of ethanol with these SCs.
However, in substance-using populations, polydrug use is
common [23]. Considerations of drug-drug interactions with
SCs are important clinically [24], but possible or unexpected
biotransformation due to co-ingested compounds should also
be considered. For example, transesteriꢀcation of cocaine
following concomitant use of ethanol and cocaine is well
known to produce the pharmacologically active metabolite
cocaethylene [25, 26]. Similarly, transesteriꢀcation has also
been demonstrated for methylphenidate, resulting in the pro-
duction of ethylphenidate (itself an NPS with misuse poten-
tial) [27]. These reactions are primarily catalysed by human
carboxylesterases (hCES) in the liver, which participate in
phase I metabolism of xenobiotics. Transesteriꢀcation of
PB-22 can also be observed as a thermal degradation artefact
when detecting by gas chromatography–mass spectrometry
Materials and methods
Chemicals and reagents
PB-22, NPB-22, 5F-PB-22 and 5F-NPB-22 were kindly pro-
vided by TICTAC Communications (London, UK). Their
identity and purity were conꢀrmed by nuclear magnetic
resonance (NMR) spectroscopy and high-performance liq-
uid chromatography–ultraviolet spectroscopy (HPLC–UV)
(% purity > 90%). Methanol, acetonitrile, formic acid (all
LC–MS grade) were purchased from Fisher Scientific
(Loughborough, UK). Triꢁuoroacetic acid (TFA), chloro-
form-d (CDCl , isotopic purity 99.8 atom %D) were from
3
Sigma-Aldrich (Dorset, UK). Deionised water was puriꢀed
1
3

Forensic Toxicology
Fig. 1 Chemical structures of
5
F-NPB-22, NPB-22, PB-22
and 5F-PB-22 (top panel), their
corresponding ethyl esters (mid-
dle panel) and their hydrolysis
product (bottom panel). SCs
synthetic cannabinoids
to 18.2 MΩ cm resistivity using a PURELAB Ultra sys-
tem (ELGA, Woodridge, UK). Aqueous phosphate buꢃer
hydrolysis products (compounds 12, 13, 16 and 17, Fig. 1)
were synthesised by alkaline hydrolysis of ethyl esters.
Summary experimental data are given in supplementary
material section S1. Ethyl esters were characterised using
(
0.5 mol/L, pH 7.4), pooled HLMs (150 donors), and
reduced nicotinamide adenine dinucleotide phosphate
NADPH) regenerating system solutions A and B were pur-
chased from Corning (Woburn, MA, USA); ethyl indazole-
-carboxylate and 1-bromo-5-ꢁuoropentane from Fluoro-
Chem (Hadꢀeld, UK); 1,5-diiodopentane from Alfa Aesar
1
13
(
NMR ( H-NMR, C-NMR, homonuclear correlation
spectroscopy—COSY, and heteronuclear single-quantum
correlation spectroscopy—HSQC) (NMR, Bruker Avance
DRX 400 MHz; Bruker BioSpin, Rheinstetten, Germany).
Further analysis was carried out using reversed-phase
high-performance liquid chromatography with diode-array
detection (HPLC–DAD, HP-1050) (Agilent Technologies,
Santa Clara, CA, USA) using a 100×2.1 mm i.d. C8 column
3
(
Lancashire, UK); ethyl indole-3-carboxylate and potas-
sium tert-butoxide (tBuOK) from Aldrich (Dorset, UK);
potassium hydroxide (KOH), bis-p-nitrophenylphosphate
(
BNPP), tetrahydrofuran (THF), hexane, ethyl acetate and
®
ethanol from Sigma-Aldrich (St. Louis, MO, USA); anhy-
drous THF from Acros Organics (Geel, Belgium).
(5 µm, Kinetex ; Phenomenex, Torrance, CA, USA). Solu-
tions of each compound [100 μg/mL in 0.1% (v/v) TFA in
water] were eluted using a water/acetonitrile gradient (both
containing 0.1% (v/v) TFA, ambient temperature, 31 min
analysis time). Finally, solutions of each compound [300 ng/
mL in 10% (v/v) acetonitrile in deionised water containing
0.3% (v/v) formic acid] were analysed by liquid chromatog-
raphy–high-resolution mass spectrometry (LC–HRMS) to
Synthesis and characterisation of ethyl esters
Synthesis of the ethyl esters (compounds 10, 11, 14 and
1
5, Fig. 1) was carried out via nucleophilic substitutions of
the N1 nitrogen atoms of (1) ethyl indazole-3-carboxylate
and (2) ethyl indole-3-carboxylate, each with 1,5-diiodo-
pentane and 1-bromo-5-ꢁuoropentane, to form the non-
2
generate reference retention times and product ion (MS )
spectra for the in vitro incubation experiments.
ꢁ
uorinated and ꢁuorinated analytes, respectively. Ester
1
3

Forensic Toxicology
In vitro incubation
immediately to 10% (over 1 min) prior to re-equilibration.
The total analysis time was 10 min.
The HLM method was based on that previously described
Heated electrospray ionisation (HESI-II) settings were:
sheath gas ꢁow-rate 70; auxiliary gas ꢁow-rate 10; spray
voltage 3.75 kV; capillary temperature 320 °C; S-lens radio
frequency level 55.0; auxiliary gas temperature 350 °C. Full-
scan acquisition parameters were: positive ionisation mode;
scan range m/z 100–1000; resolution 70,000 full width at
half maximum (FWHM); automatic gain control (AGC) tar-
[
(
19]. Brieꢁy, solutions containing 10 µg of each substrate
1 mg/mL, 10 µL) were added to Eppendorf LoBind™
tubes (Stevenage, UK), and evaporated to dryness. Buꢃer
solution (0.1 mol/L aqueous phosphate buffer, pH 7.4,
5
00 µL), NADPH-regenerating solutions [50 µL solution
+
A: 26 mmol/L NADP , 66 mmol/L glucose-6-phosphate,
6
2
and 66 mmol/L MgCl (aq); 10 µL solution B: 40 U/mL
get 1×10 . MS acquisition parameters were: parallel reac-
tion monitoring mode (based on an inclusion list containing
the appropriate precursor ions); scan range m/z 100–750;
2
glucose-6-phosphate dehydrogenase in 5 mmol/L sodium
citrate], and deionised water (390 µL) were added. The tubes
were capped, vortex-mixed, and preincubated (37 °C) with
continuous gentle shaking for 10 min using an Eppendorf
6
resolution 35,000 FWHM; AGC target 1 × 10 . The isola-
tion width was 1.2 amu. The normalised collision energy
(NCE) was optimised manually using available standards
and in-house synthesised products. For data review, total ion
chromatograms were extracted using a± 2.5 ppm extraction
window based on the exact m/z values.
®
ThermoMixer (Stevenage, UK). Reactions were initiated
by the addition of HLM solution [protein content 20 mg/mL
in 250 mmol/L sucrose (aq), 50 µL], gently mixed by hand
®
and were incubated using an Eppendorf ThermoMixer (2 h,
total reaction volume 1000 µL). A negative control was pre-
pared by incubating each of the compounds with buꢃer solu-
tion and water, omitting the HLM and NADPH solutions.
For incubations with ethanol, the initial volume of deionised
water added to the incubation mixture was reduced to 340
µL, and aqueous ethanol solution [5.86% (v/v), 50 µL] was
added. The ꢀnal concentration of ethanol was 50 mmol/L
Cannabinoid receptor assay
To assess the biological activities of the compounds, live
cell-based reporter assays that monitor protein-protein inter-
actions via the NanoLuc Binary Technology were used.
Here, the receptor activation is evaluated via the interac-
tion between β-arrestin 2 (βarr2), a cytosolic protein, and
the cannabinoid receptor CB or CB . Both βarr2 and CB /
(
0.23 g/dL). In addition, incubations were carried out in the
presence of speciꢀc carboxylesterase (CES), and nonspe-
ciꢀc esterase inhibitors (BNPP and saturated NaF solution,
respectively). For both experiments, the initial volume of
deionised water added to the incubation mixture was reduced
to 290 µL, and aqueous ethanol solution [5.86% (v/v), 50 µL]
and inhibitor solution [either 1 mmol/L BNPP, or 10% (w/v)
NaF in water, 50 μL] was added. The ꢀnal concentration of
ethanol was maintained at 50 mmol/L.
1
2
1
CB are fused to an inactive part of nanoluciferase. When
2
CB or CB are activated by a ligand, βarr2 is recruited to
1
2
the receptor, allowing interaction of the complementary
nanoluciferase subunits, yielding a functional enzyme that
generates a bioluminescent signal in the presence of the sub-
strate furimazine. Details regarding the development of the
stable CB and CB cell lines used here have been reported
1
2
After incubation, all reactions were quenched by the
addition of ice-cold acetonitrile (250 μL). Quenched solu-
tions were vortex-mixed and centrifuged (13,000 g, 15 min).
Supernatants were transferred to glass autosampler vials for
analysis by LC–HRMS (5 μL injection volume).
elsewhere [36]. The original human embryonic kidney
(HEK) T293 cell line was provided by Prof. O. De Wever
(Laboratory of Experimental Cancer Research, Department
of Radiation Oncology and Experimental Cancer Research,
Ghent University Hospital, Belgium).
The cells were routinely maintained at 37 °C, 5% CO ,
2
LC–HRMS
under humidiꢀed atmosphere in Dulbecco’s modiꢀed Eagle’s
medium (GlutaMAX™; Thermo Fisher Scientiꢀc, Waltham,
MA, USA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 100 IU/mL penicillin, 100 mg/L strep-
tomycin and 0.25 mg/L amphotericin B. Stability of the cell
lines was followed by ꢁow cytometric analysis. For experi-
ments, cells were deposited on poly-d-lysine coated 96-well
An UltiMate 3000™ HPLC system coupled to a Q
Exactive™ high-resolution mass spectrometer (both Thermo
Scientiꢀc, San Jose, CA, USA) was used. Xcalibur™ soft-
ware (version 14.0) was used for instrument control and
data analysis. Chromatographic separation was performed
using an Acquity™ BEH C18 column (50 × 2.1 mm i.d.,
4
plates (Thermo Fisher Scientiꢀc) at 5 × 10 cells/well and
1
0
.7 μm) (Waters, Borehamwood, UK). Eluents consisted of
incubated overnight. The cells were washed twice with Opti-
®
.3% (v/v) formic acid in deionised water (eluent A) and
MEM I Reduced Serum Medium (Thermo Fisher Scien-
®
in acetonitrile (eluent B). Column temperature was main-
tained at 40 °C. The elution gradient started at 10% eluent
B (2 min), ramped to 95% (over 5 min), and then decreased
tiꢀc) to remove any remaining FBS, and 100 μL Opti-MEM
I was added. The Nano-Glo Live Cell reagent (Promega,
Madison, WI, USA), a nonlytic detection reagent containing
1
3

Forensic Toxicology
the cell permeable furimazine substrate, was prepared by
Results and discussion
2
0-fold dilution of the Nano-Glo Live Cell substrate using
Nano-Glo LCS Dilution buꢃer, and 25 μL were added to
Characterisation of compounds
2
each well. Subsequently, the plate was placed into a TriStar
LB 942 multimode microplate reader (Berthold Technolo-
gies GmbH & Co., Bad Wildbad, Germany). Luminescence
was monitored during the equilibration period until the sig-
nal stabilised (15 min). We added 10 μL per well of test
compounds, present as concentrated (13.5-fold) stock solu-
Data from the characterisation experiments (NMR and
HPLC–UV) for the synthesised compounds (10-17) are
presented in supplementary material section S1. Reten-
+
tion times, precursor ion m/z values (all [M+H] ), and
major product ion m/z values for the parent compounds
and the eight synthesised compounds generated using the
LC–HRMS method are summarised in Table 1.
®
tions in 50% (v/v) methanol in Opti-MEM I. The lumi-
nescence was continuously detected for 120 min. Solvent
controls were analysed with all experiments. The ꢀnal con-
centration of methanol (3.7% v/v) has previously been veri-
For the indoles PB-22 and 5F-PB-22, the characteristic
major product ions observed were in agreement with previ-
ously published data [39], resulting from successive losses
of the hydroxyquinolinyl substructures (m/z 214.1228 and
232.1121 for PB-22 and 5F-PB-22, respectively) and the
(ꢁuoro-) alkyl chains (m/z 144.0445 and 144.0435 for both
compounds).
ꢀ
ed to be well-tolerated by the cells [36].
Curve ꢀtting and statistical analyses were performed
using GraphPad Prism software (San Diego, CA, USA).
The results are represented as mean area under the curve
(
AUC)± standard deviation (SD) with at least ꢀve replicates
for each data point (obtained in three independent experi-
Product ions spectra for the two indazoles, NPB-22 and
5F-NPB-22, showed analogous product ions (m/z 215.1183
and 233.1074 for NPB-22 and 5F-NPB-22, respectively,
plus 145.0398 and 145.0389 for both compounds). In
addition, the indazoles gave rise to fragment ions corre-
sponding to losses of the quinolinyl substructures, i.e.,
of the same m/z as the precursor ions for compounds 13
and 12, the ester hydrolysis products (m/z 233.1288 and
ments). All results were normalised to the E of JWH-018
max
(
=100%), our reference compound. Curve ꢀtting of concen-
tration-eꢃect curves via nonlinear regression (four param-
eters logistic ꢀt) was employed to determine EC (a meas-
5
0
ure of potency) and the E
(a measure of eꢂcacy). The
max
maximal eꢃect of all compounds was determined by either
the E (for the parent compounds) or the maximal eꢃect
max
that was achieved for the compound at high concentrations.
Table 1 Summary LC–HRMS data for parent and synthesised compounds PB-22, NPB-22, 5F-PB-22, 5F-NPB-22, and metabolites 10-17
Compound (formula)
RT (min) Precursor ion
accurate m/z (Δ
ppm)
Major product ion(s) accurate m/z (Δ ppm)
PB-22 (C H N O )
6.21
6.29
6.05
6.03
359.1759 (1.39) 214.1228 (0.93), 144.0445 (0.69)
360.1714 (1.94) 233.1288 (1.29), 215.1183 (1.86), 145.0398 (1.38)
377.1652 (−2.12) 232.1121 (−4.74), 144.0435 (−6.25)
23
22
2
2
NPB-22 (C H N O )
22
21
3
2
5
5
F-PB-22 (C H N O F)
23 21 2 2
F-NPB-22 (C H N O F)
378.1603 (−2.38) 251.1178 (−4.78), 233.1074 (−4.72), 213.1015 (−3.28),
22
20
3
2
1
45.0389 (−4.83)
PB-22 ethyl ester (15) (C H NO )
6.52
260.1651 (2.31) 232.1336 (1.72), 214.1231 (2.34), 188.1439 (2.66),
1
6
21
2
1
32.0811 (2.27), 118.0654 (2.54)
NPB-22 ethyl ester (11) (C H N O )
6.34
5.98
261.1598 (0.00) 233.1282 (−1.29), 215.1178 (−0.46), 145.0395 (−0.70)
278.1556 (1.80) 250.1241 (1.20), 232.1136 (1.72), 206.1345 (2.43),
1
5
20
2
2
5
F-PB-22 ethyl ester (14) (C H NO F)
16 20 2
1
32.0811 (2.27)
5
F-NPB-22 ethyl ester (10) (C H N O F)
5.82
5.61
5.21
279.1503 (0.00) 251.1188 (−0.80), 233.1083 (−0.86), 213.1021 (−0.47),
45.0396 (0.00)
232.1334 (0.86) 214.1225 (−0.47), 188.1433 (−0.53), 132.0807 (−0.76),
18.0651 (0.00)
233.1291 (2.57) 215.1183 (1.86), 163.0505 (1.84), 145.0399 (2.07)
250.1244 (2.40) 232.1135 (1.29), 206.1344 (1.94), 132.0811 (2.27)
251.1192 (2.80) 233.1084 (−0.43), 213.1022 (0.00), 145.0397 (0.69)
1
5
19
2
2
1
PB-22 ester hydrolysis product (17) (C H NO )
1
4
17
2
1
NPB-22 ester hydrolysis product (13) (C H N O )
1
3
16
2
2
5
5
F-PB-22 ester hydrolysis product (16) (C H NO F) 5.03
14 16 2
F-NPB-22 ester hydrolysis product (12)
4.85
(
C H N O F)
1
3
15
2
2
5
F-NPB-22 methyl ester (C H N O F)
5.48
265.1354 (2.64) 251.1195 (1.99), 233.1089 (1.72), 213.1029 (3.28)
1
4
17
2
2
LC–HRMS liquid chromatography–high-resolution mass spectrometry, RT retention time
1
3

Forensic Toxicology
2
5
51.1178 for NPB-22 and 5F-NPB-22, respectively). For
F-NPB-22, a deꢁuorinated product ion was also observed
hydrolysis product ions of NPB-22 and 5F-NPB-22 gave
similar results to those of their corresponding ethyl esters.
(
m/z 213.1015) which was not observed in the case of
5
F-PB-22.
HLM incubation experiments
For the ethyl ester and ester hydrolysis products of
PB-22 (compounds 15 and 17), the major protonated car-
bonyl-indole core fragment ion (m/z 144.0444) was not
observed at any signiꢀcant relative abundance. Instead,
the proposed alkyl-indole and alkyl-indole carboxylic acid
product ions (m/z 188.1439 and 232.1336, respectively)
were the major product ions for PB-22 ethyl ester, com-
pound 15 (Fig. 2), in addition to a small amount of the ion
at m/z 214.1231 also observed for both compounds 15 and
PB‑22 and 5F‑PB‑22
In the absence of ethanol, metabolism of PB-22 proceeded
as previously described to produce a series of phase I
metabolites [39]. The major metabolite (by relative abun-
dance) was the ester hydrolysis product (compound 17).
Of note, this compound was also observed at a lower
abundance (approximately 1% of the parent compound)
in the negative control samples, in which no HLMs were
included in the incubation (see supplementary material
S3.4). In addition to the ester hydrolysis product, a series
of hydroxylated metabolites were observed, with charac-
1
7. The ethyl ester of PB-22 gave rise to a product ion at
m/z 132.0811 and 118.0654, which were observed as major
product ions of PB-22 ester hydrolysis, compound 17.
With respect to the ethyl esters and hydrolysis products
of 5F-PB-22 (compounds 14 and 16), similar fragments as
PB-22 ethyl ester and ester hydrolysis were observed. The
major product ions were the alkyl-indole, m/z 206.1337;
and product ions at m/z 250.1234, 232.1129 and 118.0650
being the fragments of alkyl-indole carboxylic acid, alkyl-
indole carbaldehyde and indole group, respectively. The
ethyl ester of 5F-PB-22 also gave rise to a product ion at
m/z 132.0806.
2
teristic MS fragments suggesting monohydroxylation on
(1) the pentyl chain, (2) the indole core and (3) the quin-
olinyl substructure, carbonylation of the pentyl chain, and
successive hydroxylation products following ester hydroly-
sis and N-dealkylation. When ethanol was present, in addi-
tion to these metabolites, the previously synthesised and
characterised PB-22 ethyl ester was also detected along
with further peaks suggesting monohydroxylation of the
ethyl ester (Fig. 4). No ethyl ester was observed when
PB-22 was incubated with ethanol in the presence of either
saturated NaF (nonspeciꢀc CES inhibitor) or 1 mmol/L
BNPP (speciꢀc CES inhibitor) (see supplementary mate-
rial S3.2-S3.3). The observed LC–HRMS analyses and the
Observed product ions of NPB-22 ethyl ester and
5
F-NPB-22 ethyl ester included the alkyl-indazole carbox-
ylic acid, alkyl-indazole carbaldehyde, and indazole car-
baldehyde (m/z 233.1276, 215.1171, and 145.0391 for NPB-
2
2, m/z 251.1187, 233.1082, and 145.0395 for 5F-NPB-22,
2
respectively). The 5F-NPB-22 ethyl ester also yielded a
MS spectra of ethyl esters when SCs were incubated with
deꢁuorinated fragment (m/z 213.1020) (Fig. 3) as observed
HLM and ethanol matched with the synthetic standards
(see supplementary material S2.1-2.8 and S1.28-1.35).
2
in MS result of 5F-NPB-22 parent compound. The ester
2
Fig. 2 Product ion (MS ) spec-
trum (annotated with proposed
fragment ion structures) for the
ethyl ester of PB-22 (compound
1
5, exact m/z for protonated
molecular ion 260.1645) with
normalised collision energy
(
NCE) at 25
1
3

Forensic Toxicology
2
Fig. 3 MS spectrum (annotated
with proposed fragment ion
structures) for the ethyl ester
of 5F-NPB-22 (compound
1
0, exact m/z for protonated
molecular ion 279.1503) with
NCE at 35
Fig. 4 Overlaid extracted ion chromatograms (exact mass± 2.5 ppm)
of the major detected PB-22 human liver microsome (HLM)-derived
metabolites in the presence of ethanol: a=parent drug PB-22;
b=monohydroxylation; c=carbonylation; d=monohydroxylation
with N-dealkylation; e=ester hydrolysis (17); f=ester hydrolysis
with monohydroxylation; g=PB-22 ethyl ester (15); h = PB-22 ethyl
ester with monohydroxylation
A summary of the observed in vitro phase I metabolism,
including transesteriꢀcation in the presence of ethanol, for
PB-22 is shown in Fig. 5.
S3.8) were observed in addition to the metabolites seen for
PB-22. In the presence of ethanol, and ethanol plus CES-
inhibitor, similar results to its non-ꢁuorinated analogue were
observed.
For the ꢁuorinated indole analogue 5F-PB-22, oxida-
tive, reductive deꢁuorination and oxidative deꢁuorination
to carboxylic acid products (see supplementary material
These data demonstrate for the first-time evi-
dence of hCES-mediated transesterification of selected
1
3

Forensic Toxicology
Fig. 5 Proposed metabolic
pathway for PB-22, inclusive
of ethyl ester formation in the
presence of ethanol. (a–h: refer
to Fig. 4)
ester-containing SCs in the presence of ethanol, which gives
rise to a new family of potential markers for SC and etha-
nol concomitant use. In the case of PB-22 and 5F-PB-22,
our ꢀndings suggested that the ethyl ester formation was
catalysed by hCES (most likely hCES-1 subtype as in the
case of cocaine metabolism), because no ethyl ester forma-
tion was observed in the absence of the HLMs, nor when
CES activity was inhibited (by either nonspeciꢀc or speciꢀc
inhibitors). Furthermore, multiple hydroxylated metabolites
of the ethyl esters were also observed, which may constitute
additional useful (likely urinary) biomarkers of combined
SC/ethanol use in toxicological analyses. However, PB-22
contains a labile ester bond in the structure. Thus, when
using GC–MS with an elevated temperature in the presence
of ethanol as solvent, transesteriꢀcation to produce the ethyl
ester of PB-22 can be observed [28].
In the presence of ethanol, the ethyl esters of NPB-22
and 5F-NPB-22 were both detected. Interestingly, a small
amount of the ethyl ester was observed (approximately
0.5% of the parent compound by abundance) in the pres-
ence of only buꢃer and ethanol (i.e., without any HLMs,
see supplementary material S3.11 and S3.14). Indazole
SCs clearly display more reactivity towards nucleophilic
agents than their indole counterparts. Indeed, the NPB-22
family was more rapidly prone to hydrolysis, methanolysis
and, therefore, ethanolysis (Fig. 8), even in the absence of
any biocatalyst.
Incubation of ester hydrolysis products with HLM
To further elucidate the mechanism of formation of the
newly detected SC ethyl esters, hydrolysed SC esters (com-
pounds 12, 13, 16 and 17) were separately incubated with
HLMs to monitor any ethyl ester product formation. This
only yielded hydroxylated metabolites (data not shown).
Incubation with HLMs and ethanol did not result in the
formation of ethyl esters (illustrated in supplementary
material S4). We concluded that the investigated biocata-
lysed reactions were highly dependent on the nature of the
substrate: SC-ethanol transesteriꢀcation occurred; how-
ever, esteriꢀcation of the hydrolysed esters with ethanol
was not observed. Indeed, hCES is well known to accom-
modate ester, thioester, carbamate, and amide bonds in its
active site [40–42], suggesting that the hydrolysed esters,
bearing a free carboxylic acid, are not good substrates for
this enzyme family.
NPB‑22 and 5F‑NPB‑22
As for the two indazoles, NPB-22 produced similar in vitro
metabolites to 5F-NPB-22. Hydrolysis of the ester linkage
was the major metabolic pathway for both indazole ana-
logues, NPB-22 and 5F-NPB-22. A series of monohydroxy-
lated ester hydrolysis products were observed. In addition,
ester hydrolysis followed by oxidative deꢁuorination was
detected for 5F-NPB-22. Extracted ion chromatograms for
the metabolites of 5F-NPB-22 from in vitro HLM experi-
ments are presented in Fig. 6 and the proposed metabolic
pathway of 5F-NPB-22 (without ethanol added) is presented
in Fig. 7. To the best of our knowledge, this is the ꢀrst report
of the in vitro metabolism of 5F-NPB22.
1
3

Forensic Toxicology
Fig. 6 Overlaid extracted ion chromatograms (exact mass± 2.5 ppm) of 5F-NPB-22 HLM-derived metabolites (without ethanol): a=parent drug
5
F-NPB-22; b=ester hydrolysis (12); c=ester hydrolysis with monohydroxylation; d=ester hydrolysis with oxidative deꢁuorination
Fig. 7 Proposed metabolic
pathways for 5F-NPB-22 (a–d
refer to Fig. 6)
Biological evaluation of PB‑22, NPB‑22, 5F‑PB‑22,
5F-NPB-22) showed a concentration dependent response in
both the CB and CB receptor activation assays (Fig. 9).
5
F‑NPB‑22, their ethyl esters and their ester
1
2
hydrolysis products
The EC and E values were determined as a measure of
50 max
relative potency and eꢂcacy, respectively (see supplemen-
All parent compounds (PB-22, NPB-22, 5F-PB-22,
tary material S5.1). Both the EC and E values of PB-22
50
max
1
3

Forensic Toxicology
Fig. 8 Overlaid extracted ion chromatograms (exact mass± 2.5 ppm) of 5F-NPB-22 in the presence of methanol and ethanol (without HLM);
a=5F-NPB-22; b=hydrolysis of 5F NPB-22; c=methanolysis of 5F-NPB-22; d=ethanolysis of 5F-NPB-22
Fig. 9 Concentration-dependent response at CB (a) and CB (b) upon stimulation with diꢃerent SCs. Data are given as mean receptor activa-
1
2
tion (%)± standard deviation (SD) (n=5–8), normalised to the E of JWH-018
max
and 5F-PB-22 were in good correspondence with the previ-
ously reported result using the same reporter assays [43].
The ester hydrolysis products and ethyl esters were
also evaluated at both CB receptors (except the 5F-PB-22
hydrolysis product as this was not available for analysis,
but has previously been reported to have no activity at CB
1
and CB [43]). At CB receptors, hydrolysis resulted in a
2
1
strong reduction of the activity (to<1% that of JWH-018)
(Fig. 10a, exact values in supplementary material S5.2). The
ethyl esters of PB-22 and 5F-PB-22 also showed a strongly
1
3

Forensic Toxicology
Fig. 10 The maximal eꢃect
obtained at CB (a) and CB
1
2
(
b). Bars assigned with an
asterisk are not signiꢀcantly
diꢃerent from basal levels. Data
are given as the mean percent-
age CB receptor activation (in
comparison to the E of JWH-
max
0
18)± SD (n=5–8). EtOH
ethanol
reduced CB activation (3.2 and 4.2% for the PB-22 and
compared to the E of the parent compounds, the activity
1
max
5
F-PB-22 ethyl esters, respectively). No signiꢀcant CB
of the ester hydrolysis products did not exceed 4.73%, while
that of the ethyl esters did not exceed 28.6%. Because of the
limited receptor activation at high concentrations, the contri-
bution of these ethyl esters to the pharmacological proꢀle of
1
receptor activation was found for the indazole (NPB) deriv-
atives. When compared with the E
of the parent com-
max
pounds, the relative activity of the ester hydrolysis products
did not exceed 0.16%, while that of the ethyl esters did not
exceed 1.1%.
SCs is expected to be minimal. This is distinct from the CB
1
and CB receptor activation by the hydroxyl-metabolites of
2
With respect to CB activity, hydrolysis of the ester in the
(5F)-PB-22 (hydroxy-group on the pentyl side chain), where
2
parent compound resulted in a strong reduction of the activ-
much higher activities (>100% E JWH-018) were found
max
ity: only 6% CB receptor activation for the ester hydrolysis
[43].
2
product of PB-22 was found and an almost complete loss in
CB receptor activation was found for the ester hydrolysis
2
products of NPB-22 and 5F-NPB-22 (Fig. 10b, exact values
Conclusions
in supplementary material S5.3). For PB-22, the ethyl esters
resulted in a reduced CB activation compared to the par-
Polydrug use is very common amongst illicit and recrea-
tional drug users. Here, we have shown in vitro evidence of
the formation of SC-ethyl esters through an hCES-catalysed
transesteriꢀcation. Given that there are a number of reports
which describe fatal and nonfatal cases in which toxico-
logical ꢀndings are indicative of combined use of SCs and
2
ent compound, although still a 35.5% CB receptor activa-
2
tion (relative to JWH-018) was found at high concentrations
(
1 µM). The ethyl ester of 5F-PB-22 resulted in a 31.7%
CB activation. For the ethyl esters of the NPB-derivatives,
2
1
1.7 and 11% CB receptor activation was found. When
2
1
3

Forensic Toxicology
ethanol, it is suggested that clinical and forensic laborato-
ries should monitor the potential appearance of the reported
ethyl esters and/or their hydroxylated metabolites to facili-
tate interpretation of such cases. It is worth mentioning that
detection of the described ethyl esters may be associated
with several ester-containing SCs (common metabolites to a
number of SCs); therefore, their detection should be accom-
panied by the concomitant presence of parent drugs (often
diꢂcult to be detected in urine) or appropriate metabolites
that are unique to a certain SC to avoid any ambiguous
reporting. The elimination proꢀles are needed to understand
their potential contribution to toxicity in humans using SCs,
especially if they prove to have a longer half-life than the
parent compound. However, the ethics of administration of
these unlicensed substances to humans makes this diꢂcult,
and the relevance of animal studies is questionable.
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the provision of SC standards. The authors acknowledge the Royal Thai
Government Scholarship for supporting this work. Annelies Cannaert
and Christophe Stove acknowledge the support from Ghent University-
Special Research Fund (BOF; PDO026-18 and BOF project Grants No.
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Compliance with ethical standards
(
PMID:27257717)
Conflict of interest Paul Dargan is a member of the Scientiꢀc Commit-
tee of the European Monitoring Centre for Drugs and Drug Addiction
and Chair of the Scientiꢀc Committee of the European Association of
Poisons Control Centres and Clinical Toxicologists. All other authors
have no conꢁict of interest to declare.
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Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
1
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Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
1
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