Synthesis of methoxetamine, its metabolites and deuterium labelled analog as analytical standards and their HPLC and chiral capillary electrophoresis separation

Article abstract of DOI:10.1039/c7ra10893a

Methoxetamine, a designer drug marketed as a replacement for the dissociative anaesthetic ketamine, has been associated with significant numbers of hospital related intoxications and deaths in Europe. The fast and user-friendly identification and quantification of methoxetamine and its metabolites is a key factor for successful treatment of intoxication. Therefore, we suggested a convenient preparation method which was used for the synthesis of methoxetamine, seven methoxetamine metabolites and a deuterium labelled derivative as analytical standards. Methoxetamine and normethoxetamine were used as starting materials for the preparation of O-demethylated and N-dealkylated metabolites. The multistep synthesis starts from commercially available compounds and offers good yields. Our prepared analytical standards were used for the confirmation of the suggested structure of methoxetamine metabolites in rat urine by LCMS. Capillary electrophoresis was used for the chiral separation of MXE and its metabolites using β-cyclodextrin, carboxymethylated β-cyclodextrin, and sulphated β-cyclodextrin as chiral selectors at various concentrations. Chiral separation was successful for four analytes. A mixture of MXE and its metabolites was subsequently analyzed under optimal conditions, i.e. when using 15 mmol L^-1 β-cyclodextrin in 50 mmol L^-1 phosphate buffer, pH 2.5. In this case, chiral separation was achieved for three analytes and all analytes were separated from each other.

Full text of DOI:10.1039/c7ra10893a

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Synthesis of methoxetamine, its metabolites and
deuterium labelled analog as analytical standards
Cite this: RSC Adv., 2017, 7, 56691
and their HPLC and chiral capillary electrophoresis
separation†
ab
B. Jurasek,
M. Himl,c R. Jurok,bc K. Hajkova,bde A. Vobinuskova,d P. Rezankad
abe
*
and M. Kuchar
Methoxetamine, a designer drug marketed as a replacement for the dissociative anaesthetic ketamine, has
been associated with significant numbers of hospital related intoxications and deaths in Europe. The fast
and user-friendly identification and quantification of methoxetamine and its metabolites is a key factor
for successful treatment of intoxication. Therefore, we suggested a convenient preparation method
which was used for the synthesis of methoxetamine, seven methoxetamine metabolites and a deuterium
labelled derivative as analytical standards. Methoxetamine and normethoxetamine were used as starting
materials for the preparation of O-demethylated and N-dealkylated metabolites. The multistep synthesis
starts from commercially available compounds and offers good yields. Our prepared analytical standards
were used for the confirmation of the suggested structure of methoxetamine metabolites in rat urine by
LCMS. Capillary electrophoresis was used for the chiral separation of MXE and its metabolites using
b-cyclodextrin, carboxymethylated b-cyclodextrin, and sulphated b-cyclodextrin as chiral selectors at
various concentrations. Chiral separation was successful for four analytes. A mixture of MXE and its
metabolites was subsequently analyzed under optimal conditions, i.e. when using 15 mmol Lꢀ1
b-cyclodextrin in 50 mmol Lꢀ1 phosphate buffer, pH 2.5. In this case, chiral separation was achieved for
three analytes and all analytes were separated from each other.
Received 2nd October 2017
Accepted 9th December 2017
DOI: 10.1039/c7ra10893a
rsc.li/rsc-advances
growing popularity of these compounds is based on availability
and apparent ‘legality’ since many NPSs are yet to be covered by
Introduction
legislative controls pertaining to narcotic and psychotropic
substances.3–5 In 2016, 66 NPS were notied to the EU Early
Warning System operating under EMCDDA. The total number
of monitored substances increased to more than 620.4 The NPS
market is rapidly growing and its legislative regulation is very
difficult. In addition to the large number of NPS notied every
year, there is a very limited supply (oen complete absence) of
analytical standards that are important for research, clinical
and forensic practice. EMCDDA divides NPS into several
classes.6 Dissociative anaesthetics are an interesting, however
dangerous, group of NPS. The dissociative anaesthetics include
compounds with an aryl moiety connected to the cyclohexyl-
amine (arylcyclohexylamines). Arylcyclohexylamines are repre-
sented by ketamine and its analogues (Fig. 1).
According to the annual drug reports published by the Euro-
pean Monitoring Centre for Drugs and Drug Addiction
(EMCDDA), a new class of psychoactive substances, known as
‘legal highs’, ‘herbal incense’, ‘designer drugs’ or ‘research
chemicals’ has emerged on the drug market in the last decade.
The new psychoactive substances (NPSs) include a wide range of
products, from natural plant-originating substances to semi-
synthetic and synthetic compounds.1,2 In recent years, the use of
new psychoactive substances has grown dramatically. The
a
´
Department of Chemistry of Natural Compounds UCT Prague, Technicka 5, Prague,
166 28, Czech Republic. E-mail: kuchara@vscht.cz
b
´
Forensic Laboratory of Biologically Active Substances UCT Prague, Technicka 5,
Prague, 166 28, Czech Republic
Ketamine is a well-known anaesthetic with a good safety
prole, which is used particularly in anaesthesia, and as co-
analgesics in pain management. Moreover, ketamine is sug-
gested as a novel treatment for major depressive disorder and
bipolar depression.7,8 On the other hand, ketamine (2), phen-
cyclidine (PCP, 1) and methoxetamine (MXE, 3) have an abuse
potential.9–11 While ketamine (2) has been registered for
c
´
Department of Organic Chemistry UCT Prague, Technicka 5, Prague, 166 28, Czech
Republic
d
´
Department of Analytical Chemistry UCT Prague, Technicka 5, Prague, 166 28, Czech
Republic
e
´
National Institute of Mental Health CZ, Topolova 748, Klecany, 250 67, Czech
Republic
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra10893a
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standards are particularly effective for mass spectrometry
analyses and quantication. Many studies have been reported
for the preparation of deuterium-labelled controlled drugs as
internal standards for the use in GCMS and LCMS analysis.25,26
Therefore, our aim was to develop a convenient method for
the synthesis of methoxetamine, its metabolites and deuterium-
labelled derivative as analytical standards. All prepared high
purity compounds were used for pharmacokinetics.10,27
Fig. 1 Phencyclidine (1), ketamine (2), methoxetamine (3).
Chiral analysis of drugs and their metabolites can provide
pharmaceutical usage, many less well characterized arylcyclo- very useful information about metabolic pathways in living
hexylamines have appeared on the black market as ‘research organisms because it can be expected that metabolic pathways
chemicals’ and can be obtained from internet retailers.11,12
can be different for each enantiomer. Thus enantiomers may
Methoxetamine (3) is a structural analogue of ketamine. have different pharmacological effects. For this reason we used
MXE was extensively advertised for recreational use as a ‘legal’ capillary electrophoresis (CE) as chiral separation method for
and ‘bladder friendly’ alternative to ketamine with the main enantioseparation of MXE and its metabolites using cyclodex-
effects of hallucinations, depersonalization and dissociation of trin and their derivatives as chiral additives to the background
the physical body.13–16 MXE has a sub-micromolar affinity for the electrolyte (BGE).
glutamate N-methyl-D-aspartate (NMDA) receptor (pKi ¼ 6.59),
which is comparable to that of ketamine (pKi ¼ 6.18), and binds
Results and discussion
Synthesis of methoxetamine and its metabolites
to the phencyclidine site on the NMDA receptor.17 Very recently,
we have published that methoxetamine behaves as a standard
dissociative anaesthetic more potent than its analogue ket-
amine, which in turn might increase the risk of overdose, loss of
consciousness and potentially fatal complications. MXE was
found to be generally anxiogenic and psychotomimetic, which,
in humans is likely to produce unpleasant experiences, and
negative psychological sequelae.10
Data on the prevalence of use of methoxetamine (3) is
limited, but reports of acute intoxications including fatalities
have recently appeared.18–20 Methoxetamine is associated with
120 non-fatal intoxications and 22 deaths (reported through
Early warning system (EWS) by European Member States),
however only 55 of these cases have been analytically
conrmed.15,21 Although many related publications have
focused on intoxication with methoxetamine, complete
synthesis have not been published yet. Only a brief study of
methoxetamine analysis reported the possibility of methoxet-
amine synthesis analogously to ketamine.22
The synthetic route to obtain N-dealkylated methoxetamine
metabolites is outlined in Fig. 2. Following the synthetic route,
treating commercially available 3-methoxybromobenzene (4)
with cyclohexanone and activated magnesium under the
Grignard protocol afforded intermediate 5,28 which under acidic
conditions eliminated water to cyclohexene intermediate 629 in
high yield 90%. Following epoxidation underwent smoothly in
biphases system CH2Cl2/H2O without phase transfer catalyst to
obtain the epoxide 7.30 Regioselective opening of the epoxide
with HBr (aq) gave unstable bromohydrine, which was directly
oxidized with Jones reagent to afford compound 8 in 64%
overall yield. Nucleophilic substitution of 8 with NaN3 was
Toxicological detection is necessary as a proof of intoxication
or abuse of a certain compound. Urine analysis is one of the
most common detection methods in forensic toxicology. Urine
samples are usually analysed because of longer detectability,
however the knowledge about the metabolism of the investi-
gated compound is oen essential, especially, if the substance
is completely metabolised and only metabolites are detectable
in the urine samples. Furthermore, knowledge about metabo-
lism is a prerequisite for the risk assessment concerning
pharmacokinetic interactions caused, e.g. by cytochrome P450
(CYP) enzymes. The rst structures of methoxetamine metab-
olites have been suggested by Meyer et al. and Menzies et al.23,24
They used full-scan GCMS (EI) technique, but this determina-
tion has some limitations. Therefore, we synthetized standards
of methoxetamine metabolites for the conrmation of their
suggested structures and for the pharmacokinetic and metab-
olism studies. To support the pharmacokinetic and metabolism
studies of methoxetamine, stable isotopically labelled stan-
Fig. 2 Synthesis of N-dealkylated metabolites 10, 11, 13 and deute-
dards are an essential tool. Deuterium-labelled internal rium labelled metabolite 14.
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followed by almost quantitative azide
9
reduction using analysis; the rst one was the common co-elution of 10 and its
catalytic hydrogenation. The metabolite normethoxetamine 10 deuterium-labelled analog 14 and the second one was caused by
was treated with NaBH4 affording dihydro-normethoxetamine 11 similar retention of 3 and 11, which differ in m/z. The rst stage
in average yield 65%. The synthesis of metabolite dihydro-O- of separation was accomplished successfully and the second
desmethyl-normethoxetamine 13 started from normethoxet- stage was to create sensitive multiple reaction monitoring
amine 10 through intermediate O-desmethyl-normethoxetamine method (MRM) for MS/MS acquisition. Source conditions and
12 obtained by methoxy group cleavage with HBr (aq) reux fol- mass transitions were optimized for each analyte. Mixtures of
lowed by reduction with NaBH4 in high 85% overall yield. O- analyzed compounds were measured and evaluated on different
Desmethyl-normethoxetamine (12) was selectively protected with concentration levels, and subsequently several different
di-tert-butyldicarbonate and then methylated with CD3I. Final matrices were used (listed in Fig. 4.).10,27
acid-promoted deprotection provided deuterium labelled
metabolite 14.
However, chiral separation of the analytes was not pursued
in the aforementioned publications. In addition, there is no
A synthetic route for the preparation of O-demethylated information on the metabolic pathways for particular enantio-
metabolites (Fig. 3) was led through the parent compound mers. Thus, it is important to develop not only chromatographic
methoxetamine (3). We followed the methoxetamine synthesis separation method, but, in particular, a method that can be
described by Hays.22 Briey, 3-methoxybenzonitrile 15 under- used to differentiate between the enantiomers of a particular
went Grignard reaction to form 3-methoxyphenyl cyclopentyl drug or its metabolite.
ketone (1631). Further bromination led to alpha-bromo ketone
17,32 which was treated with ethylamine to form Schiff's base
and nal heating provided methoxetamine (3).22 Metabolite
CE analysis of prepared standards
dihydro-O-desmethyl-methoxetamine (21) was obtained by
methoxy group cleavage with HBr (aq) under reux followed by
reduction with NaBH4 in high overall yield (65%). Direct
reduction of methoxetamine with NaBH4 afforded dihy-
dromethoxetamine metabolite 19 in high yield (96%).
Our CE study dealt with a suitable chiral selector, its concen-
tration, and pH of BGE for the enantiomers separations of
studied MXE and its metabolites. Three selected cyclodextrins
LC-MS/MS analysis of prepared standards
A sample preparation technique with an LC-MS/MS detection
method using synthesized standards was developed and
extensively evaluated before it was used for the real samples of
pharmacokinetic study. Full description of this method and
collected data is shown in our previous work.10,27
The aim of the chromatography was to separate compounds
with similar m/z so that there was no misidentication because
of isobar occurrence. There were just two co-elutions within the
Fig. 4 Chromatograms of matrix-matched calibration on 50 ng mLꢀ1
(approx. 0.2 mmol Lꢀ1) concentration level. Different tissues – liver,
Fig. 3 Synthesis of methoxetamine (3) and N-demethylated metab- brain, and lung – were enriched by standard compounds and prepared
olites 19, 20, 21.
in the same way as the real samples.
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were tested at all above mentioned conditions, i.e. at 4 different
concentration levels and 6 different pH. For b-cyclodextrin
(BCD) that is oen used due to low cost, its chiral separation
was achieved for compound 3 (R ¼ 0.8) and metabolites 11 (R ¼
0.6) and 20 (R ¼ 1.1). The second tested cyclodextrin was car-
boxymethylated b-cyclodextrin (CMBCD) that is oen used for
separation of compounds containing basic nitrogen group. In
this case, compound 3 was partially enantioseparated (R ¼ 1.0)
and metabolites 10, 11, and 20 were fully enantioseparated (R >
1.5) (Fig. 5). Using sulphated b-cyclodextrin (SBCD) did not lead
to the better resolution, although the same previously
mentioned four compounds were also at least partially sepa-
rated (0.5 < R < 1.5). To our best knowledge, we achieved
enantioseparations of these compounds by CE for the rst time.
Because in the living organisms above-mentioned
compounds will occur simultaneously, the second part of our
CE study focused on the separation of mixtures of MXE and its
metabolites. The measurement was carried out under optimal
conditions that were chosen from experiments at above-
mentioned conditions, i.e. 3 types of cyclodextrins, 4 different
concentration levels and 6 different pH. The optimal BGE
consists of 50 mmol Lꢀ1 phosphate buffer (pH 2.5) with
15 mmol Lꢀ1 BCD as a chiral additive.
Fig. 6 Electropherogram of analyzed mixture of MXE and its metab-
olites; dependences of absorbance at 207 nm on electromigration
time; BGE: 15 mmol Lꢀ1 BCD, 50 mmol Lꢀ1 sodium phosphate, pH 2.5;
fused-silica capillary: od/id
¼
375/75 mm, total/effective length
58.5/50.0 cm; voltage 20 kV; temperature 25 ꢁC.
compound. As can be seen in Fig. 6, six analytes were separated
from each other and the half of them was partially
enantioseparated.
Conclusions
To identify individual signals in the electropherogram as
depicted in Fig. 6, new mixtures were prepared with various
concentrations of studied chiral compounds. These mixtures
were analysed under the identical conditions as the original
mixture and the assignment of the signals was carried out using
the peak areas and UV-vis spectra of individual analysed
In conclusion, we established an efficient synthetic route
towards normethoxetamine-d3 (14) and seven methoxetamine
metabolites, namely, dihydro-O-desmethyl-normethoxetamine
(13), O-desmethyl-normethoxetamine (12), O-desmethylme-
thoxetamine (20), dihydro-O-desmethylmethoxetamine (21),
normethoxetamine (10), dihydro-normethoxetamine (11),
dihydromethoxetamine (19).
A sample preparation technique with LC-MS/MS method
using synthesized standards was developed and extensively
evaluated before it was used on the real samples of pharmaco-
kinetic study. Mixtures of analyzed compounds were measured
and evaluated on different concentration levels, and subse-
quently several different matrices were also used. Full descrip-
tion of this method and collected data is shown in our previous
work.10,27
We studied the inuence of the type and concentration of
CDs on the enantioselectivity of methoxetamine and its deriv-
atives by chiral CE. The results of the analyses (carried out in
50 mmol Lꢀ1 buffer at pH 2.5 to 5.0 with the addition of 4 to
15 mmol Lꢀ1 BCD, CMBCD or SBCD) demonstrate a signicant
dependence of enantioseparations on the conditions studied.
Four compounds (3, 10, 11, and 20) were successfully
enantioseparated.
Experimental section
Chemicals and materials
Fig. 5 Electropherograms of analyzed compounds; dependences of
absorbance at 207 nm on electromigration time. Separated analytes Hydrochloric acid (30%) (Suprapur, Merck, Germany),
(BGE): (A) 3 (10 mmol Lꢀ1 CMBCD, 50 mmol Lꢀ1 phosphate buffer, pH
1 mol Lꢀ1 sodium hydroxide (Tripur, Merck), ortho-phosphoric
2.5); (B) 10 (10 mmol Lꢀ1 CMBCD, 50 mmol Lꢀ1 formic buffer, pH 4.0);
acid (50%), b-cyclodextrin (BCD), carboxymethylated b-cyclo-
(C) 11 (10 mmol Lꢀ1 CMBCD, 50 mmol Lꢀ1 acetic buffer, pH 5.0); (D) 20
dextrin (CMBCD) and sulphated b-cyclodextrin (SBCD) (all
Sigma-Aldrich, Czech Republic). Diethyl ether solution of
hydrochloride was prepared from commercial diethyl ether,
(7 mmol Lꢀ1 CMBCD, 50 mmol Lꢀ1 formic buffer, pH 4.0); fused-silica
capillary: od/id ¼ 375/75 mm, total/effective length 58.5/50.0 cm;
voltage 20 kV; temperature 25 ꢁC.
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commercial concentrated hydrochloric acid and calcium
chloride as drying agents. Solvents were acquired from
commercial sources and were used aer distillation. Other
commercial reagents were used without further purication.
The reactions were monitored with the aid of thin-layer
chromatography (TLC Silica gel 60 F254) and pre-coated
reverse phase gel plates (TLC RP-18 F254). Visualization was
carried out with UV light. Chromatography refers to ash
column chromatography and it was carried out with the
indicated solvents on silica gel (particle size 0.040–0.060 mm)
or reverse phase column (Redisep RF Gold C18). All operations
involving air-sensitive reagents were performed under an
inert atmosphere of dry argon using syringe and cannula
techniques, oven-dried glassware, and freshly distilled and
dried solvents. Thermal rearrangement were carried out in
Microwave reactor (Biotage Initiator Classic) melting points
were determined using a melting point apparatus (PGH Run-
dfunk–Fernsehen), and they are uncorrected. NMR spectra
were recorded in CDCl3 or CD3OD at room temperature on
a Varian Gemini 300 MHz (1H NMR 300 MHz, 13C NMR
75 MHz) or an Agilent 400 MR DDR2 (1H NMR 400 MHz, 13C
NMR 100 MHz). The spectra were referenced to residual
solvent protons in the 1H NMR spectra (7.26 ppm for
CDCl3 and 4.84 and 3.31 ppm for CD3OD) and to solvent
carbons in the 13C NMR spectra (77.0 ppm for CDCl3 and
49.05 ppm for CD3OD). Infrared (IR) spectra were measured on
FTIR Nicolet 6700 (Thermo-Nicolet) coupled with ATR cell
GladiATR (Pike). High-resolution MS (HRMS) were run either
by the ESI ionization or atmospheric pressure chemical
ionization (APCI) mode. High resolution mass spectra
(HRMS) were recorded using a Thermo Scientic LTQ Orbitrap
Velos.
CE conditions
Individual analyzed compounds were dissolved in water at
10 mmol Lꢀ1 concentration. For the CE experiments the analyte
solutions were further diluted with water to the nal concen-
tration 1 mmol Lꢀ1. For the determination of migration order of
particular compounds the mixture composed of analyzed
compounds at different concentration was used. The back-
ground electrolyte consisted of 50 mmol Lꢀ1 buffers at pH 2.5,
3.0 (50 mmol Lꢀ1 orthophosphoric acid adjusted to appropriate
pH with 1 mol Lꢀ1 NaOH), 3.5, 4.0 (50 mmol Lꢀ1 formic acid
adjusted to appropriate pH with 1 mol Lꢀ1 NaOH), 4.5, 5.0
(50 mmol Lꢀ1 acetic acid adjusted to appropriate pH with
1 mol Lꢀ1 NaOH), and various concentrations of the studied
CDs (4, 7, 10, 15 mmol Lꢀ1 for BCD, CMBCD, and SBCD). A new
fused silica capillary was rst rinsed with 1 mol Lꢀ1 NaOH for
30 min, then with H2O for 30 min. Between the runs the
capillary was rinsed at 99.4 kPa rst with 0.1 mol Lꢀ1 NaOH for
2 min then with H2O also for 2 min, and nally with running
buffer again for 2 min (for the capillary washing a different
buffer solution than for the subsequent analysis was used). The
analytes were injected hydrodynamically by pressure of 1.5 kPa
for 5 s. All the separations were performed at 20 kV (anode at the
injection capillary end) with a voltage ramp time of 12 s.
Detection was carried out at 207 nm and the capillary was
ꢁ
thermostated at 25 C during the analyses.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
CE separations were performed with an Agilent CE instru-
ment (Agilent 3D HPCE, Germany) equipped with UV-vis diode-
array detector. Bare fused silica capillary of 375/75 mm od/id and
58.5/50 cm total/effective length obtained from Polymicro
Technologies (AZ, USA) was used.
This study was funded by a specic university research (project
MSMT No. 20-SVV/2017) and by the Ministry of Interior of the
Czech Republic (project VG20172020056).
Notes and references
LC conditions
LC-MS/MS was performed using an Agilent 1200 HPLC series
(Agilent Technologies, Germany) in tandem with API 3200 MS
system (AB Sciex, Canada). ZORBAX Eclipse Plus C18 (50 ꢂ 2.1
mm, 5 mm) with a pre-column was used for the chromato-
graphic separation with a gradient of 0.1% (v/v) formic acid in
deionized water (mobile phase A) and 0.1% (v/v) formic acid
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