Synthesis, absolute configuration and in vitro cytotoxicity of deschloroketamine enantiomers: rediscovered and abused dissociative anaesthetic

Article abstract of DOI:10.1039/c8nj03107j

In this study, we aim to determine differences in cytotoxicity of racemic deschloroketamine and its enantiomers. The synthesized racemate of this recently rediscovered and abused dissociative anaesthetic was resolved by chiral HPLC and the absolute configuration of the enantiomers was assigned using a combination of circular dichroism methods and single-crystal X-ray. Not only the absolute configuration, but also the most preferred conformers present in the crystal were successfully determined by electron and vibrational circular dichroism supported by ab initio calculations, and confirmed by X-ray. The in vitro cytotoxicity of racemic deschloroketamine and its enantiomers was determined for nine different types of cell lines. Generally, (S)-deschloroketamine exhibited higher cytotoxicity in the majority of cases. For human embryonic kidney cells (HEK 293T), the (S)-enantiomer reached the IC₅₀ below 1 mM concentration and, in consequence, proved to be twice as potent as the (R)-enantiomer. On the other hand, live-cell fluorescence microscopy imaging at sub-IC₅₀ concentrations provided evidence for only a minor effect of deschloroketamine racemate and enantiomers on endoplasmic reticulum stress and mitochondria morphology in neuroblastoma cells SH-SY5Y.

Full text of DOI:10.1039/c8nj03107j

Volume 42 Number 24 21 December 2018 Pages 19309–20250
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New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
ISSN 1144-0546
PAPER
Martin Kuchař, Michal Kohout et al.
Synthesis, absolute configuration and in vitro cytotoxicity of
deschloroketamine enantiomers: rediscovered and abused dissociative
anaesthetic

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Synthesis, absolute configuration and in vitro
cytotoxicity of deschloroketamine enantiomers:
rediscovered and abused dissociative anaesthetic†
Cite this: New J. Chem., 2018,
42, 19360
ab
c
d
e
ˇ
Frantisek Kralık,‡ Silvie Rimpelova, Jan Cejka,
ˇ ´´ ´
Bronislav Jurasek,
‡
´
Vladimır Setnicka,c Tomas Ruml,d Martin Kuchar*ab and Michal Kohout
´ˇ
*
f
´
ˇ
ˇ
In this study, we aim to determine differences in cytotoxicity of racemic deschloroketamine and its
enantiomers. The synthesized racemate of this recently rediscovered and abused dissociative anaesthetic was
resolved by chiral HPLC and the absolute configuration of the enantiomers was assigned using a combination
of circular dichroism methods and single-crystal X-ray. Not only the absolute configuration, but also the most
preferred conformers present in the crystal were successfully determined by electron and vibrational circular
dichroism supported by ab initio calculations, and confirmed by X-ray. The in vitro cytotoxicity of racemic
deschloroketamine and its enantiomers was determined for nine different types of cell lines. Generally,
(S)-deschloroketamine exhibited higher cytotoxicity in the majority of cases. For human embryonic
kidney cells (HEK 293T), the (S)-enantiomer reached the IC50 below 1 mM concentration and, in
consequence, proved to be twice as potent as the (R)-enantiomer. On the other hand, live-cell
fluorescence microscopy imaging at sub-IC50 concentrations provided evidence for only a minor effect
of deschloroketamine racemate and enantiomers on endoplasmic reticulum stress and mitochondria
morphology in neuroblastoma cells SH-SY5Y.
Received 22nd June 2018,
Accepted 26th October 2018
DOI: 10.1039/c8nj03107j
rsc.li/njc
groups of recreational drugs represent cathinones, synthetic
cannabinoids, phenethylamines, tryptamines, piperazines and
Introduction
The prevalence of new psychoactive substances (NPS) that are arylcyclohexylamines. Among these substances, there are many
not controlled under existing legislation is a complex phenom- pharmaceuticals previously approved for clinical use and sub-
enon affecting the health and safety of citizens on the global level. sequently discontinued or even pharmaceuticals that are still
According to the European Monitoring Centre for Drugs and Drug being used in human or veterinary medicine.2–4
Addiction (EMCDDA) 2017 update on NPS in Europe, more than
A dissociative anaesthetic ketamine (1) was introduced to
620 NPS are currently being monitored.1 The most important the market in 1970 for analgesia and sedation in clinical
practice. However, if administered at a sub-narcotic dose, it
a Forensic Laboratory of Biologically Active Substances, University of Chemistry and
can cause hallucinations, floating sensations, dissociation
without the actual loss of consciousness, therefore, it is often
misused as a recreational drug.5–8 Nightmares, changes in
perception, flash-backs, tolerance and dependence as well as
chronic changes in the urinary tract are frequently reported
adverse effects.7,9 These adverse effects have led to a progressive
change in the ‘‘psychonauts’’ and ‘‘clubbers’’ communities, which
recently started to prefer methoxetamine (2) – a more potent disso-
ciative anaesthetic (marketed as ‘‘bladder-friendly’’ temporarily legal
alternative to ketamine).10 Additionally, several novel dissociative
anaesthetics with ketamine-like effects, such as deschloroketamine
(3), ephenidine (4) and lefetamine (5) (Fig. 1) have recently been
introduced and are being used as ketamine replacements.11,12
Deschloroketamine, an analogue of ketamine, is not a new
substance.13,14 Originally, it was proposed as an immuno-
modulation agent and for the treatment of bacterial, fungal,
´
Technology Prague, Technicka 5, 166 28, Prague 6, Czech Republic.
E-mail: martin.kuchar.vscht.cz
b Department of Chemistry of Natural Compounds, University of Chemistry and
´
Technology Prague, Technicka 5, 166 28, Prague 6, Czech Republic
c Department of Analytical Chemistry, University of Chemistry and Technology
´
Prague, Technicka 5, 166 28, Prague 6, Czech Republic
d Department of Biochemistry and Microbiology, University of Chemistry and
´
Technology Prague, Technicka 3, 166 28, Prague 6, Czech Republic
e Department of Solid State Chemistry, University of Chemistry and Technology
´
Prague, Technicka 5, 166 28, Prague 6, Czech Republic
f Department of Organic Chemistry, University of Chemistry and Technology Prague,
´
Technicka 5, 166 28, Prague 6, Czech Republic. E-mail: michal.kohout@vscht.cz
† Electronic supplementary information (ESI) available: Details on synthesis and
characterization of racemic deschloroketamine, preparative enantiomer separa-
tion and single-crystal X-ray parameters is available. CCDC 1838630–1838632. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c8nj03107j
‡ Contributed equally.
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Scheme 1 Synthesis of deschloroketamine (3).
Fig. 1 Structures of ketamine (1), methoxetamine (2), deschloroketamine
(3), ephenidine (4) and lefetamine (5).
Since no data were available on enantioseparation of
deschloroketamine, an analytical high-performance liquid
chromatographic (HPLC) separation method was developed
and subsequently transferred to preparative scale HPLC (for
details, see ESI†). The racemic substance was separated into
pure enantiomers (Fig. 2) and the purity of collected fractions
was verified in the analytical mode (inset of Fig. 2).
viral or protozoan infections.15,16 It has, however, never been
put in the clinical practice. Recently, it has been rediscovered as
a ‘‘legal’’ alternative to ketamine and is now being abused in a
similar manner. According to the anecdotal reports of users, it
is about 1.5 times more potent than methoxetamine and about
three times more potent than ketamine,17 which makes it a real
threat to health and safety of the users. This is, in particular,
due to the lack of information on deschloroketamine pharmaco-
kinetics, pharmacodynamics, its toxicity as well as the toxicity of its
metabolites. In addition, there is no information available on
difference in the effect of different stereoisomers of this drug.
Therefore, we have performed a pilot study with this highly
potent drug addressing the important features of chirality.
We have synthesized racemic deschloroketamine and separated
its enantiomers using high-performance liquid chromatography
(HPLC) on a chiral polysaccharide column. Furthermore, we have
established the absolute configuration of the enantiomers using a
powerful combination of vibrational circular dichroism supported
by ab initio calculations and X-ray diffraction.18 Subsequently,
in vitro cytotoxicity of both enantiomers was studied and related
to the toxicity data known for ketamine.19,20 Such information is
essential for the potential use of deschloroketamine in human
medicine, especially, analogously to ketamine, as a rapid acting
antidepressant.
Determination of the absolute configuration of enantiomers
Spectroscopic and ab initio methods
Conformational analysis. Because most physico-chemical
properties are shared by both enantiomers and their geometries
are mirror images, it is sufficient to perform geometry optimiza-
tion only for one of them, in this case the (R)-enantiomer. The key
question for successful conformational search is the position of
the phenyl and methylammonium (–+NH2CH3) group, which are
located at the same carbon atom of the cyclohexane ring. In order
to fully cover the conformational possibilities, two basic structural
models were created: model A with the phenyl group in the
axial position and the –+NH2CH3 group in the equatorial
position of the cyclohexane ring, and model B with switched
positions of the groups.
For each model, values of dihedral angles defining the
mutual orientation of the phenyl and –+NH2CH3 groups were
systematically varied in order to produce 12 geometries as
starting points for further optimization. The structures were
optimized using density functional theory (DFT) approach at
Results
Synthesis and enantioseparation
Deschloroketamine (3) was synthesized in accord with pre-
viously published methods (Scheme 1).13,14 First, benzonitrile
reacted with cyclopentyl magnesium bromide in diethyl ether
for 72 h giving rise to ketone 7. Due to the instability of the
intermediates 8 and 9, it is advisable to accomplish the next
synthetic steps in a one-pot manner. Regioselective bromina-
tion of cyclopentyl phenyl ketone (7) provided bromoketone 8,
which was used without further purification in the reaction
with methylamine to afford hydroxy imine 9. Again, after
evaporating the unreacted species, the crude imine 9 was
subjected to a microwave-assisted thermal rearrangement in
decalin to provide deschloroketamine (3). The crude product
was stabilized in the form of hydrogen chloride salt and
purified by multiple crystallizations to provide chemically pure
substance, which was subjected to chiral separation.
Fig. 2 Preparative enantioseparation of deschloroketamine with marked
areas of the collected mobile phase. The inset shows verification of the
enantiopurity of combined fractions of peak 1 (DCK-1) and peak 2 (DCK-2)
in an analytical mode, which was found 499% for both enantiomers.
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CAM-B3LYP/aug-cc-pVDZ and oB97X-D/TZVP levels of theory
using Gaussian 09 program package.21,22
The DFT optimization of the starting structures yielded
3 stable conformers for the model A and 4 stable conformers
for the model B (for their relative Gibbs free energies and
corresponding Boltzmann weights, see ESI,† Table S1). The
calculated energies of all 3 conformers of the model A (i.e. with
the phenyl group in the axial position) were significantly lower
than those of the model B. This result was slightly unexpected,
as it was assumed that the rather bulky phenyl unit should be
located in the equatorial position due to lower steric hindrance.
However, after closer inspection, the resulting conformers of
the model A are stabilized by a hydrogen bond between one
hydrogen of the ammonium group and the oxygen atom of the
carbonyl group.
According to the Boltzmann weights, only two conformers of
the model A seemed to be relevant for further simulations of
the spectra, with conformer I being predominantly populated
(Fig. 3, left). The major difference between the geometries
of the conformations lays in the orientation of the –+NH2CH3
group (see ESI,† Fig. S2 and Table S1).
Fig. 4 Experimental VCD spectra of DCK-1 and DCK-2 samples (top)
compared with the calculated spectra for (R)-DCK (bottom).
intensities in the middle part of the spectra (1700–1350) were
lower, the spectral profile in this area was also very well
predicted by the calculation.
Assignment of absolute configuration of deschloroketamine
enantiomers
To confirm the results obtained with the VCD spectroscopy,
ECD spectroscopy was applied for determining the absolute
configuration of DCK enantiomers. Experimental spectra of
DCK-1 and DCK-2 (Fig. 5) show again that the previous separa-
tion of the enantiomers was successful because of the opposite
sign of the spectra. Three intense bands were observed in the
experimental spectra: a broad band with the maximum localized
approximately at 290 nm and two sharper bands localized at 220
and 194 nm (for major orbital contributions to experimentally
observed ECD bands, see ESI†). A very good agreement was found
As expected, the VCD spectra of DCK enantiomers of unknown
absolute configuration labelled as DCK-1 and DCK-2 (Fig. 4)
were of the opposite sign, confirming that two enantiomers
were present. Besides VCD spectra, also IR spectra were
recorded (ESI,† Fig. S2) as they may help to correctly assign
calculated transitions to experimentally observed bands. Only IR
and VCD spectra calculated with oB97X-D/TZVP are presented as
this method provided better results than B3LYP/6-31++G**. The
calculated spectra were scaled with the factor of 0.952, which was
derived from the CompareVOA algorithm.23
A very good agreement was found in a comparison of the
calculated VCD spectrum for (R)-DCK with the experimental
spectrum of DCK-2, in which all the intense bands were
correctly predicted by the calculations. There were two spectral
regions, in which it was possible to reliably assign the absolute
configuration: first, the region of 1750–1700 cmÀ1, where an
intense band of the valence vibration of carbonyl group is
located (negative sign of the band for (R)-DCK) and second,
the region of 1350–1280 cmÀ1, where two positive bands were
observed in the spectrum of (R)-DCK. Although the band
Fig. 5 Experimental ECD (top) spectra of DCK-1 and DCK-2 samples
compared with the calculated spectra for (R)-DCK (bottom).
Fig. 3 Geometries of two lowest-energy conformers of (R)-DCK.
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Table 1 Evaluation of in vitro cytotoxicity of deschlorketamine and its
enantiomers
between the calculated ECD spectrum for (R)-DCK and the experi-
mental spectrum of DCK-2, which corresponds with the results
obtained from VCD measurements.
Cell line
Hep HEK
Compound G2 293T
SH-
SY5Y
HMC- HT-
3 29 5637 H9C2 HaCaT L929
X-ray diffraction of hydrochloride salt of enantiomers
In order to confirm the results obtained from spectroscopic
IC50 [mM]
methods, X-ray diffraction of single crystals of (S)-DCK, (R)-DCK Rac-DCK 2.10 1.43
2.48 1.54 1.58 1.52 3.00 1.82
2.43 1.63 1.55 1.43 3.92 1.71
3.86 1.70 1.62 1.75 4.23 2.00
1.86
2.74
2.12
(S)-DCK
(R)-DCK
2.24 0.99
2.41 1.82
and racemic forms was performed (for detailed information on
crystal system, see ESI†). To this date, powder diffraction pattern of
(S)-DCK is the only available data in the literature.24 Therefore,
single crystal X-ray analysis was performed and the obtained
structures of (R)- and (S)-DCK were compared with the structures
predicted by the spectroscopic methods and ab initio calculations.
As expected, the resulting crystal structures of the enantiomers
were mirror images of each other. Surprisingly, two symmetrically
independent molecules were found in the asymmetric unit. Both
the spectroscopic methods and X-ray provided identical results.
It is interesting to note that the ab initio methods successfully
predicted the most favourable conformers of the respective
enantiomers, since both these conformers were also detected
in the crystal structures (Fig. 6).
which (S)-DCK enantiomer reached the IC50 below 1 mM
concentration. Moreover, the (S)-DCK was almost two times
more potent than (R)-DCK enantiomer. Small differences in the
cytotoxicities of individual enantiomers were observed also in
an in vitro cardiac model of H9C2 cells and SH-SY5Y cells from
neuroblastoma. The latter express opioid, muscarinic and
nerve growth factor receptors as well as exhibit neuronal
marker enzyme activity.25 Since the differences among the
racemate and individual enantiomers were in this cell line
rather marginal, we assume that similarly to ketamine,20 the
toxicity is not induced via a stereoselective receptor.
Surprisingly, the IC50 of (rac)-DCK and the enantiomers in
Hep G2 liver cells, which are a model for hepatotoxicity
studies,26 were not significantly decreased compared to other
cell lines. This might be caused by very low expression of CYP
enzymes in Hep G2 cells. Therefore, the drug metabolites,
which are usually the most toxic, cannot be probably properly
formed. Nevertheless, at high concentration, the drug may also
induce cell death, but without the relation to the CYP activity
normally present in primary hepatocytes.27 Even though Hep
G2 cells are among the most widely used for this purpose and have
many liver-specific functions, some authors have described that
they really lack functional expression of most human liver enzyme
P450s,28 or that it is ca. 2–3 times lower than in primary
hepatocytes.29
Overall, the in vitro cytotoxicity of DCK was higher than what is
described for ketamine by, e.g., Braun et al. (2010),20 Bai et al.
(2011),30 Shen et al. (2015),31 and Kovacova et al. (2016);32 although,
needless to say, the IC50 values of ketamine were determined only
at 24 and/or 48 h. In clinics, ketamine is used at 1.5 mM
concentration33 or even at 93 mM in patients with chronic
pain,34 which is concentration much higher than concentrations
already inducing toxicity in in vitro evaluations.20 The lower IC50
values of DCK in comparison to ketamine are in agreement with
Frison et al. (2016), who reported that DCK is considered to be
more potent and longer lasting than ketamine.35
In vitro cytotoxicity of deschloroketamine and its enantiomers
The in vitro cytotoxic profile of deschlorketamine and its two
enantiomers was assessed using cell metabolic activity assay
WST-1 after 72 h of the compound’s treatment. The following
cell lines were used to evaluate hepatic, renal, neuronal,
cardiac, and urinary toxicity: seven human model cell lines
Hep G2, HEK 293T, 5637, HT-29, HMC-3, SH-SY5Y, and HaCaT
(derived from liver, kidney, urinary bladder, colon, microglia,
neuroblastoma, and epidermis, respectively) as well as one rat
H9C2 and one mouse cell line L929 (derived from heart and
fibroblasts, respectively). The IC50 values of all tested cell lines
after 72 h treatment by (rac)-DCK and its enantiomers are listed
in Table 1.
The in vitro toxicity of deschlorketamine was concentration-
dependent and the IC50 values were in millimolar concentra-
tions. Interestingly, we detected significant differences in
cytotoxicities not only among individual compounds but also
across the individual cell lines. The highest cytotoxicity was
determined for human embryonic kidney cells (HEK 293T), in
Further cytotoxicity measurement using, e.g., HepaRG cells,
by their characteristics resembling primary human hepato-
cytes, and detailed mechanistic studies will be needed to
unravel the toxicity of this NPS.
Fluorescence microscopy
Based on the observations made on ketamine by Braun et al.
(2010),20 who described its significant impact on mitochondrial
function of iPSc cells already at 20 mM concentration after 24 h
Fig. 6 Fitted structures of the most abundant conformers of R-enantiomer
(DCK-2) found in the crystal using X-ray analysis (purple and blue) and the
most stable conformers predicted by ab initio calculations (ochre and red).
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treatment (no decrease in cell viability at this concentration) and the response, including differences in adverse effects.37 For ketamine,
fact that, at millimolar concentrations, ketamine induces apoptosis which we use here as a reference drug, it was reported that the
via the mitochondrial pathway independent of death receptor (S)-enantiomer is more potent than the (R)-enantiomer,3 which
signalling,20 and that 1 mM ketamine induces prolonged elevation is in agreement with its higher potency for the inhibition of
of calcium ions in cytosol triggering mitochondrial, and possibly NMDAR (N-methyl-D-aspartate receptor),38 but also its toxicity.19
also endoplasmic reticular (ER), stress,36 we have decided to The NMDAR inhibitory pathway of ketamine is also responsible for
evaluate morphology of mitochondria and endoplasmic reticulum its rapid antidepressant effect, which is vividly studied.39–42
in neuroblastoma cells SH-SY5Y at two sub-IC50 concentrations Recently it has been claimed that a major metabolite of ketamine
(250 and 500 mM) of (rac)-DCK and its enantiomers after 72 h found in human plasma, (2R, 6R)-hydroxynorketamine, acts as a
treatment by using live-cell fluorescence microscopy imaging, rapid antidepressant in mice through an unspecific activation of
see Fig. 7.
AMPAR (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
At 250 mM concentration (72 h), neither (rac)-DCK nor its receptor).43 This proposed mechanism is not completely accepted
enantiomers had impact on the morphology of mitochondria yet, since several previous as well as very recent studies point at a
and endoplasmic reticulum (data not shown). However, at significant contribution of NMDAR inhibition in the antidepressant
500 mM concentration, as it is apparent from Fig. 7, rac-DCK as well action of ketamine and its metabolites. These studies involve
as (S)-DCK and (R)-DCK treatment resulted in ER stress pronounced the racemic form, but lately pure (R)- and (S)-ketamine and
by aggregation of the ER network when compared to control their enantiomerically pure metabolites, in particular (2R, 6R)-
untreated cells (Fig. 7) and cells treated only by the vehicle. Impact hydroxynorketamine and (2S, 6S)-hydroxynorketamine, seem to
of DCK treatment on morphology of mitochondrial network was not play the main role.43–47 Currently, it seems that the long-lasting
apparent at sub-IC50 concentration of 500 mM, nevertheless, we have antidepressant function of ketamine is induced by the hydroxy-
observed weakened mitochondrial staining in DCK ((S)-DCK, norketamine metabolites. However, since a long-term treat-
(R)-DCK) treated cells compared to the control. Similar observation ment, even with very low doses of ketamine, may result in the
was made for ketamine-treated neuron-like cells stained by Mito- development of the above-mentioned adverse effects,19,20 alter-
Tracker Red, the fluorescence intensity of which was decreased.20
native drugs with higher potency are required to diminish this
risk for patients.
Deschloroketamine seems to be an appealing alternative to
ketamine, since, according to the anecdotal reports of clubbers
and psychonauts, it is 2–3 times more potent than ketamine.
This is highly favourable, as deschloroketamine may be most
probably administered in much lower doses than ketamine. In
addition, it seems to harm the urinary track to a lower extent
than ketamine, which may also be beneficial for potential
medical application, despite slightly higher toxicity we report
here. Considering the enantiomerically pure compounds, the
R-enantiomer can be more suitable for potential use in medicine,
since it is less toxic than the S-enantiomer. Assuming similarity in
the pharmacological profile of deschloroketamine and ketamine,
the most active deschloroketamine metabolite i.e. (2R, 6R)-
hydroxynordeschloroketamine may also exert rapid and sustained
antidepressant activity as well as possess no ketamine-related side
effects.43,47
Discussion
In general, each enantiomer can undergo a different metabolic
pathway in the living system, thus, providing different biological
Conclusions
We have synthesized and characterized rac-deschloroketamine
and developed a chiral HPLC method to harvest the pure
enantiomers, (R)- and (S)-deschloroketamine. The absolute
configuration of the single enantiomers has been determined
by a combination of ab initio calculated VCD and ECD spectra
and their comparison with experimental results. The results
have been confirmed by single-crystal X-ray structure analyses of
hydrochlorides of both enantiomers. The most stable conformers
predicted by calculations have also been found in single crystal
structures, which documents high reliability of the chosen combi-
nation of spectroscopy with ab initio calculation methods. Similarly
Fig. 7 Live-cell fluorescence microscopy of SH-SY5Y cells treated with
deschlorketamine (rac-DCK) and its enantiomers ((S)-DCK and (R)-DCK) at
500 mM concentration for 72 h. (A, E, I, M) bright field images; (B, F, J, N)
endoplasmic reticulum labelled by ER-Tracker Blue-White DPX; (C, G, K, O)
mitochondria stained with a mitochondria-specific dye based on a penta-
methinium salt; (D, H, L, P) merge.
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to ketamine, (S)-deschloroketamine exhibits higher cytotoxicity and subsequent column chromatography (eluent hexane/ethyl
than the (R)-enantiomer. However, the observed toxicity is most acetate, 95/5). It was obtained 6.9 g (26%) of a clear liquid.
probably not induced via a stereosensitive receptor. In addition, 1H NMR, d: 1.58–1.80 (m, 4H), 1.87–1.97 (m, 4H), 3.72 (m, 1H),
pathogenic changes on the morphology of mitochondria and 7.46 (m, 2H), 7.54 (m, 1H), 7.97 (m, 2H). 13C NMR, d: 202.9 (CQO),
endoplasmic reticulum have not been observed at 250 mM 137.1 (Ar-Cq), 132.8 (Ar-CH), 128.63 (Ar-CH), 128.58 (Ar-CH), 46.5
concentrations.
(CH), 30.1 (2 Â CH2), 26.4 (2 Â CH2).
In conclusion, based on our results, the toxicity of deschloro-
1-Bromocyclopentyl phenyl ketone (8). A solution of ketone 2
ketamine can be considered similar to ketamine. These results (6.7 g, 38.5 mmol) in dry dichloromethane (30 mL) in the inert
are highly promising, since deschloroketamine is more potent argon atmosphere was titrated with a solution of bromine
and its effects are longer lasting than those of ketamine. Such (3.1 g, 38.5 mmol) in dry dichloromethane (10 mL) until the
properties may be highly beneficial for the potential medical use red colour of the mixture did not disappear. Then the reaction
of deschloroketamine, therefore, its antidepressant effect should mixture was stirred for 30 min at room temperature. The
be thoroughly investigated.
unreacted solvent and the traces of residual bromine were
evaporated in vacuum using 2 M sodium hydroxide solution
at the outlet of the apparatus. The crude product 8 was
obtained as a yellowish liquid (8.1 g, 32.0 mmol) in 83% yield,
which was immediately used in the next reaction step. 1H NMR,
d: 1.73–1.88 (m, 2H), 1.99–2.15 (m, 2H), 2.36–2.58 (m, 4H), 7.45
(m, 2H), 7.54 (m, 1H), 8.15 (m, 2H). 13C NMR, d: 195.4 (CQO),
134.9 (Ar-Cq), 132.9 (Ar-CH), 130.4 (Ar-CH), 128.3 (Ar-CH), 71.3
(Cq), 41.1 (2 Â CH2), 23.6 (2 Â CH2).
Experimental section
Chemicals used for the synthesis were obtained from commercial
sources (Sigma Aldrich, TCI Europe, Acros Organics). Diethyl ether
solution of hydrochloride was prepared from commercial diethyl
ether, commercial concentrated hydrochloric acid and calcium
chloride as a drying agent. Solvents were acquired from commer-
cial sources and were used after distillation. Solvents denoted as
dry, were dried before use by molecular sieves. Other commercial
reagents were used without further purification. 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). All operations involving air-sensitive reagents were per-
formed under an inert atmosphere of dry argon and dried solvents.
Thermal rearrangement was carried out in a microwave reactor
(Biotage Initiator Classic). Melting points were determined using a
melting point apparatus (PGH Rundfunk–Fernsehen), and they are
uncorrected. Solvents used for analytical and preparative enantio-
separation were of standard HPLC grade and were purchased
from VWR International. NMR spectroscopy was performed on
an Agilent 400-MR DDR2 spectrometer operating at 400.13 MHz
for 1H and 100.62 MHz for 13C. Chemical shifts were referenced
internally to the signal of the residual solvent. 2D NMR spectra
were obtained using standard sequences as supplied by the
manufacturer.
Deschloroketamine hydrochloride (3). Ketone 8 (8.1 g,
32.0 mmol) was under cooling (0 1C) dissolved in methylamine
(10 mL) and the reaction mixture was stirred in the inert argon
atmosphere for 2 h. Unreacted methylamine was evaporated
and the residue was suspended in diethyl ether (30 mL). The
solids were filtered off, the filtrate was evaporated and the
obtained hydroxy imine 9 was immediately dissolved in decalin
(10 mL). The solution was stirred and heated in a microwave
reactor to 190 1C for 4 h. The reaction mixture was cooled to
room temperature and diluted with a solution of hydrogen
chloride in diethyl ether (4 mL). The precipitate was filtered,
washed with acetone and purified via multiple crystallizations
from a methanol/diethyl ether mixture. Deschloroketamine
hydrochloride 3.0 g (12.5 mmol, 30%) was isolated as a white
1
powder, m.p. 215–216 1C. H NMR, d: 1.69–1.93 (m, 2H), 1.94–
2.21 (m, 3H), 2.34 (s, 3H), 2.39–2.63 (m, 2H), 3.21–3.37 (m, 1H),
7.49 (m, 2H), 7.62 (m, 3H). 13C NMR, d: 207.2 (CQO), 131.9
(Ar-CH), 131.3 (Ar-CH), 131.0 (Ar-Cq), 129.4 (Ar-CH), 73.0 (Cq),
40.1 (CH2), 32.8 (CH2), 28.6 (CH2), 27.2 (CH3), 22.8 (CH2).
Synthetic procedures
Enantioseparation procedure
Cyclopentyl phenyl ketone (7). To a cold (0 1C) solution of Analytical enantioseparation of deschloroketamine was per-
benzonitrile (16.0 g, 0.16 mol) in diethyl ether (50 mL), a formed using Agilent 1100 series system equipped with a diode
solution of cyclopentylmagnesium bromide (26.0 g, 0.15 mol) array detector, binary pump, column thermostat, degasser and
in diethyl ether (75 mL) was added dropwise in the inert argon an autosampler. Sample concentration was 1 mg mLÀ1, the flow
atmosphere. The mixture was stirred for 1 h at 0 1C, the cooling rate was set to 1 mL minÀ1 and the temperature was kept at
bath was removed and the reaction mixture was stirred at room 23 1C (temperature was set to match the room temperature at
temperature for 72 h. Then it was decomposed with a few drops which the preparative separation was performed). Measure-
of a saturated aqueous ammonium chloride solution and ments were carried out on a chiral polysaccharide column
subsequently acidified with aqueous 1 M hydrochloric acid. ChiralArt Amylose SA.
The layers were separated and the aqueous layer was extracted
Preparative scale enantioseparation was carried out using
with diethyl ether (2 Â 50 mL). The collected organic solution was AutoPurification System (Waters) equipped with a binary
washed with water (2 Â 20 mL) and dried with anhydrous pump, column manager, diode array detector and fraction
magnesium sulphate. The solvent was evaporated and the crude collector. The enantioseparation was performed on a chiral
product was purified by vacuum distillation (95–99 1C/0.5 Torr) column Chiralpak IA 250 Â 21 mm ID, 5 mm, which contained
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the same type of a chiral selector as the analytical column, v2015;49 unit cell refinement: APEX3 v2015; data reduction:
namely tris[3,5-dimethylphenylcarbamoyl]amylose immobilized SAINT.49 The structure was solved by direct methods in SIR92;50
on the 5 mm silica support. The sample concentration was full matrix least squares refinement on F2 was provided by
10 mg mLÀ1, the flow rate was set to 15 mL minÀ1
.
CRYSTALS.51
The X-ray CIF files for the structures have been deposited at
the Cambridge Crystallographic Data Center with the deposi-
Spectroscopic measurements
For all electronic circular dichroism (ECD) measurements, tion numbers CCDC 1838630–1838632.†
solutions of deschloroketamine hydrochloride were used in
All H atoms were located in a difference map, but reposi-
demineralised water with a concentration of 7 mg LÀ1. The tioned geometrically, then, they were initially refined with soft
ECD spectra were recorded by J-815 spectrometer (Jasco, Japan) restraints on the bond lengths and angles to regularize their
in a quartz cuvette (Hellma, Germany) with the optical path geometry (C–H in the range of 0.93–0.98 Å, N–H set to 0.86 Å,
length of 1 cm at room temperature. The measurement condi- and O–H to 0.82 Å) and Uiso(H) (in the range 1.2–1.5 times Ueq
tions were set as follows: a spectral region of 185–350 nm, of the parent atom), after which the positions of carbon bound
scanning speed of 20 nm minÀ1, response time of 8 s, resolution of hydrogen atoms were refined with riding constraints. The same
0.5 nm, band width of 1 nm, and sensitivity of 100 mdeg. The final set of soft restraints was used during the final stages of the
spectra were obtained as an average of 3 accumulations, which refinement, when the positional parameters of the hetero
were corrected for the baseline by subtracting the spectra of bound hydrogen atoms were refined. Absolute configuration
demineralised water under the same experimental conditions.
For all infrared spectroscopy (IR) and vibrational circular
dichroism (VCD) measurements, solutions of deschloroketamine
was determined by the refinement of Flack’s x parameter.
Evaluation of in vitro cytotoxicity
hydrochloride in D2O (ISOSAR deuterierte Produkte GmbH, Cytotoxicity of deschlorketamine and its R- and S-enantiomer
Germany, at least 99.8% of D) with a concentration of 100 g LÀ1 was evaluated in vitro by a WST-1 viability assay (Sigma, USA),
were used at room temperature. The IR and VCD spectra were based on reduction of tetrazolium salt (WST-1 substrate) into
recorded in a spectral region of 1750–1275 cmÀ1 using FT-IR soluble formazan by mitochondrial oxidoreductases in meta-
IFS 66/S spectrometer equipped with PMA 37 VCD/IRRAS bolically active cells. The following human cell lines were used
module (Bruker, Germany). The samples were measured in for this assay: cells from hepatocellular carcinoma (Hep G2),
BioCell (Biotools, USA) with CaF2 windows and the optical path cells from colorectal adenocarcinoma (HT-29), keratinocytes
length of 23 mm. The VCD spectra were recorded with the (HaCaT), embryonic kidney cells (HEK 293T), cells from urinary
resolution of 4 cmÀ1 and they are presented as an average of bladder grade II carcinoma (5637), microglial cells (HMC3),
7–9 blocks, each consisting of 2260 scans.
cells from neuroblastoma (SH-SY5Y); one rat cell line derived
from heart (H9C2), and one mouse cell fibroblast cell line
(L929). The cells were seeded into individual wells of 96-well
Computational methods
For all computations, the solute–solvent interactions were plates (10 000 cells per well) in 100 mL of cell culture medium
considered via the conductor-like continuum model implemented recommended by ATCC (American Tissue Culture Collection)
within the polarisable continuum model48 with a default dielectric supplemented with 10% fetal bovine serum (FBS). After 16 h of
constant for water of e = 78.3553. Boltzmann populations, needed incubation at standard cell cultivation conditions (37 1C, 5%
for computing the weighted average of spectra of the lowest-energy CO2, 95% humidity), the cells were influenced with the tested
conformers, were derived from relative Gibbs free energies at a compounds at final concentrations of 0–5 mM in 100 mL of
temperature of 298 K.
fresh cell cultivation media with 10% FBS. After 72 h, the cell
IR and VCD spectra were calculated at the oB97X-D/TZVP culture medium was removed and fresh 100 mL of cell culture
level. The IR and VCD spectra were visualized using Lorentzian medium without phenol red with 10 mL of WST-1 was added.
band profile with a bandwidth at half-maximum of 6 cmÀ1
.
Then, after 2 h incubation, the absorbance of formed formazan
Transition frequencies were scaled in order to improve the was measured spectrophotometrically at 450 nm (the reference
similarity of theoretical and experimental spectra23 and to wavelength of 630 nm) using UV-vis spectrometer (BioRad).
ensure the correct assigning of corresponding bands.
The absorbance is directly proportional to the oxidoreductase
ECD spectra were calculated at the CAM-B3LYP/aug-cc-pVDZ activity, and thus to the number of metabolically active cells. All
levels. For visualization of the calculated spectra, Gaussian samples were tested in quadruplicates. Cells treated only with
band profile was used. Bandwidths were optimized for indivi- cell culture medium and cells treated with a vehicle (dimethyl
dual bands in order to increase similarity between calculated sulfoxide and methanol) served as controls. The IC50 values
and experimental spectra.
were determined as the concentration necessary to kill
50% of cells.
Crystallographic study
Fluorescence microscopy
X-ray structure analysis was carried out on a Bruker D8
VENTURE system equipped with a multilayer monochromator, SH-SY5Y cells (1 Â 105 per well) were seeded into 35 mm glass
a Cu Ka Incoatec microfocus sealed tube (l = 1.54178 Å) using bottom dishes for live-cell imaging (Ibidi plates, Germany) and
combined f and o scans at 180 K. Data collection: APEX3 left to adhere overnight (ca. 16 h). The attached cells were
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NJC
washed with pre-warmed phosphate buffered saline (PBS, pH 7.4, Acknowledgements
37 1C) and incubated with deschloroketamine or its enantiomers
This research was supported by Czech Science Foundation (project
No. 16-17689Y), Ministry of Interior of the Czech Republic (MV0/
VI20172020056), JIGA 2017 UCT grant and Specific University
Research (project 20-SVV/2017 and 21-SVV/2018).
(0.1, 0.25 and 0.5 mM concentration) dissolved in complete cell
culture medium without phenol red (FluoroBrite DMEM, Thermo
Fisher Scientific) for 72 h. Stock solutions of the tested compounds
were prepared in methanol, which was always used as a control.
After the incubation period, the cells were washed with PBS. Then,
cell organelles, such as mitochondria and endoplasmic reticulum
were stained with a mitochondria-specific dye (50 nM, 10 min),52
and a commercial marker ER-Tracker Blue-White DPX (Thermo
Fisher Scientific; 120 nM, 30 min). After the incubation period,
the cells were washed with PBS and fed with fresh phenol
red-free medium. The impact of deschloroketamine on cell
morphology and physiology of cell organelles was studied by real-
time live-cell fluorescence microscopy at 37 1C and in 5% CO2
atmosphere. The images were acquired by an inverse fluorescence
microscope Olympus IX-81 equipped with xCellence system (high-
stability 150 W xenon arc burner, EM-CCD camera C9100-02,
60Â oil immersion objective with NA 1.4) and by OMX Blaze
microscope (Softworx, sCMOS camera, 60Â oil immersion objective).
Notes and references
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Abbreviations
5637
Urinary bladder cells
AMPAR
a-Amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid receptor
B3LYP
Becke, three-parameter, Lee–Yang–Parr
exchange correlation functional
Coulomb attenuated method
Cytochrome P450 enzymes
Deschloroketamine
CAM
CYP
DCK
DFT
ECD
EMCDDA
Density functional theory
Electron circular dichroism
European monitoring centre for drugs and
drug addiction
ESI
Electronic supplementary information
Mouse cell line derived from heart cells
Epidermis cells
Embryonic kidney cells
Liver cells
Microganglia cells
High-performance liquid chromatography
Colon cells
Half maximal inhibitory concentration
Mouse fibroblast cells
H9C2
HaCaT
HEK 293T
Hep G2
HMC3
HPLC
HT-29
IC50
L929
oB97X
Dual-corrected exchange–correlation
functional
NMDAR
NPS
SH-SY5Y
VCD
N-Methyl-D-aspartate receptor
New psychoactive substances
Neuroblastoma cells
Vibrational circular dichroism.
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Conflicts of interest
The authors declare no competing financial interest.
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