Structural spectroscopic study of enantiomerically pure synthetic cathinones and their major metabolites

Article abstract of DOI:10.1039/d0nj05065b

New psychoactive substances (NPSs) have become a popular alternative to illicit drugs of abuse. However, to determine their metabolic pathways in the human organism, a detailed knowledge of their structure is crucial. Here, we present a comprehensive spectroscopic structural study of synthetic cathinones (clephedrone, flephedrone, and brephedrone) and their major human metabolites, desmethyl derivatives. Chiral high-performance liquid chromatography was utilized to obtain the individual enantiomers of the parent synthetic cathinones and their assumed major metabolites synthesized de novo. The developed chromatographic method made it possible to obtain the target optically pure substances on a multimilligram scale. Electronic and vibrational circular dichroism, combined with infrared and ultraviolet spectroscopy and supported by DFT calculations, were used to determine their absolute configuration and the chiroptical methods to elucidate their molecular structure in detail. Two stable conformers of each substance were found in aqueous solution. Their relative abundances were estimated based on the Boltzmann distribution and the population weighted spectra were obtained. Very good agreement was achieved between the experimental and simulated spectra, enabling the 3D structures of the studied substances to be determined in aqueous solution. This journal is

Full text of DOI:10.1039/d0nj05065b

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DOI: 10.1039/D0NJ05065B
Structural spectroscopic study of enantiomerically pure synthetic
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cathinones and their major metabolites
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Dita Spálovská*a, Martin Paškanb, Bronislav Jurásekc, Martin Kuchařc, Michal Kohoutb, Vladimír
Setničkaa
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a
Department of Analytical Chemistry, University of Chemistry and Technology, Prague 6, Czech
Republic
b
Department of Organic Chemistry, University of Chemistry and Technology, Prague 6, Czech
Republic
c
Forensic Laboratory of Biologically Active Substances, Department of Chemistry of Natural
Compounds, University of Chemistry and Technology, Prague 6, Czech Republic
Abstract
New psychoactive substances (NPSs) have become a popular alternative to illicit drugs
of abuse. However, to determine their metabolic pathways in the human organism, a detailed
knowledge of their structure is crucial. Here, we present a comprehensive spectroscopic
structural study of the synthetic cathinones (clephedrone, flephedrone, brephedrone) and their
major human metabolites, desmethyl derivatives. Chiral high-performance liquid
chromatography was utilized to obtain the individual enantiomers of the parent synthetic
cathinones and their assumed major metabolites synthesized de novo. The developed
chromatographic method made it possible to obtain the target optically pure substances on a
multimilligram scale. Electronic and vibrational circular dichroism, combined with infrared and
ultraviolet spectroscopy and supported by DFT calculations, were used to determine their
absolute configuration and the chiroptical methods elucidate their molecular structure in detail.
Two stable conformers of each substance were found in aqueous solution. Their relative
abundances were estimated based on the Boltzmann distribution and population weighted
spectra were obtained. Very good agreement was achieved between the experimental and
simulated spectra, enabling the 3D structures of the studied substances to be determined in
aqueous solution.
Keywords
Synthetic cathinones, conformation, chiroptical spectroscopy, DFT calculations,
preparative enantioseparation, chiral high-performance liquid chromatography
1

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DOI: 10.1039/D0NJ05065B
1 Introduction
New psychoactive substances (NPSs) are synthetic drugs of abuse that mimic either the
structure or effects or both the structure and effects of illicit drugs, such as cocaine,
amphetamine, methylenedioxymethamphetamine (MDMA) or tetrahydrocannabinol (THC).
With their stimulatory effects, synthetic cathinones are among a widening group of NPSs. They
are derivatives of (S)-(−)-cathinone, a psychoactive substance naturally occurring in the plant
Catha edulis. The growing popularity of NPSs generally, and cathinones particularly, is
connected with social and health problems, resulting in many of them becoming banned.
Despite the high prevalence, their toxicology and pharmacology in the human organism is not
fully understood yet. Furthermore, it is reasonable to assume that cathinone enantiomers may
evince different psychoactive and toxicological properties like amphetamines,1, 2 synthetic
cannabinoids3, 4 or dissociative anaesthetics.5, 6 Hence, not only their physiological effects, but
also pharmacological profile including the metabolism of the enantiomers of these substances
in the human organism remain to be uncovered. However, to enable the studies of chiral
substance properties, methodology must be developed to obtain chemically and optically pure
enantiomers, which may be further optimized for the analysis of biological samples.
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The enantiomers can be obtained either via direct synthesis7, 8 or chiral resolution
(predominantly by crystallization9 or separation technique10, 11 on a preparative scale). Chiral
separation of cathinones on an analytical scale has already been achieved by a number of
different methods (including gas chromatography, liquid chromatography, capillary
electrophoresis, etc.) summarized in a recent review.10 Currently, the most widely employed
technique for the chiral separation of NPSs remains high-performance liquid chromatography
(HPLC).12 This is because it enables not only the analytical screening of newly introduced
NPSs,13-18 but also their chiral separation on the semi-preparative and preparative scales. This
allows a subsequent detailed study of the exact molecular structure of the drug18-20 and possibly
even its pharmacological properties.5, 21
The absolute configuration of enantiomers obtained in this way can be experimentally
determined by single crystal X-ray diffraction (XRD).22 Such crystallographic characterization
of the structures of some cathinone derivatives has already been performed for several NPSs,
including
1‑(4‑fluorophenyl)‑2‑(methylamino)pentan‑1‑one
(4‑FPD)
and
1‑(4‑methylphenyl)‑2‑(ethylamino)pentan‑1‑one (4‑MEAP),23 as well as metaphedrone and
pentedrone.24 However, the XRD requires a single crystal of the respective substances, and in
addition the compound conformation in a single crystal may considerably differ from the
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conformation of the substance in solution and in vivo. For this reason, it may be DbOeIn: 1e0f.1i0c3i9a/lD0tNoJ05065B
assign the absolute configuration of a chiral substance or to distinguish individual enantiomers
from each other by chiroptical spectroscopy.25-28 Chiroptical spectroscopy [electronic circular
dichroism (ECD) and vibrational circular dichroism (VCD)] in combination with ab initio
calculations29, 30 already demonstrated its benefits in several studies determining the detailed
structure of several cathinones,19, 20 cannabinoids3, 4 or a dissociative anaesthetic5 in a solution.
However, much work remains to be done as many NPSs have yet to be spectroscopically
characterized and are yet to have their detailed structures in solution determined.
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In this article, we present comprehensive analytical methods for chiral analyses of three
synthetic cathinones (flephedrone, clephedrone, brephedrone) and their expected desmethyl
metabolites (norflephedrone, norclephedrone, norbrephedrone) in aqueous solution. The
analytical separation method was further upscaled into convenient semipreparative mode usable
for physical separation of the mentioned cathinones. Moreover, to elucidate and compare the
3D structures of the NPSs enantiomers, VCD, ECD, IR and ultraviolet (UV) absorption
spectroscopy were used, supported by DFT calculations at the B3LYP/6-311++G(d,p) level of
theory, including solvent effects. The absolute configuration of the studied substances were
assigned and their 3D structures revealed. Furthermore, the expected major metabolites
(desmethyl derivatives) were characterized by ECD spectrocopy.
2 Materials and methods
2.1 Chemicals
Standards of flephedrone, clephedrone and brephedrone hydrochlorides were purchased
from Alfarma (Czech Republic) with a purity of > 98%. Norflephedrone, norclephedrone and
norbrefedrone hydrochlorides were synthesized in house (for details, see Electronic
Supplementary Information – ESI). Deuterium oxide (99.9% D) was purchased from ISOSAR
GmbH, Germany. HPLC grade solvents, heptane, hexane, propan-2-ol (2-PrOH), and ethanol
(EtOH) were obtained from Merck (Prague, Czech Republic). Diethyl ether was purchased from
Sigma Aldrich (Prague, Czech Republic) and diethyl ether (p.a. grade) from Lach-ner
(Neratovice, Czech Republic). Diethyl ether was dried over sodium and distilled before use.
The solution of dry hydrogen chloride in diethyl ether was prepared in-house.
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2.2 Chiral separation
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The analytical scale method for the chiral separation of the studied substances was
developed using Agilent 1100 series system (Agilent Technologies, Waldbronn, Germany)
using a polysaccharide-based chiral stationary phase Chiral ART Amylose-SA (250×4.6 mm,
id, 5 µm) (YMC, Europe, Dinslaken, Germany). The sample concentration was 1 g∙l−1 and the
injection volume was set to 10 μl. The mobile phase screening was performed at a flow rate of
1 ml∙min−1; a wavelength of 254 nm was used for analyte detection. Preparative
enantioseparation was performed on the AutoPurification System (Waters, Milford, MA, USA)
using a polysaccharide-based preparative column Chiralpak IA (250×20 mm, id, 5 µm) from
Chiral Technologies Europe (Illkirch, France).
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The samples of flephedrone, clephedrone and brephedrone were prepared as follows:
the drug (30 mg) was dissolved in 2-PrOH (2 ml) containing 1% DEA, and the solution was
diluted with heptane 5 ml. The samples of norflephedrone, norclephedrone and norbrephedrone
were prepared by dissolving the compounds (50 mg) in a solvent mixture consisting of EtOH/2-
PrOH/hexane (2.5/1.0/2.5 ml) with DEA (50 μL). The solutions were filtered through a 0.4 μm
PTFE syringe filter (Labicom, Olomouc, Czech Republic) and subjected to preparative chiral
separation (vide infra). Fractions containing the individual enantiomers of flephedrone,
clephedrone and brephedrone were collected, evaporated to dryness and the residual solvent
traces (particularly DEA) were removed under vacuum (30 min) at room temperature. The
obtained yellowish oils were dissolved in diethyl ether and filtered. The obtained solutions were
treated with dry hydrogen chloride in diethyl ether to provide hydrochloride salts of the single
enantiomers of drugs as crystalline material, which were evaporated on rotavap and
subsequently dried under vacuum (4.0·10−3 bar). Fractions containing the individual
enantiomers of nor-derivates were treated with hydrogen chloride in diethyl ether, solutions
evaporated on rotavap, and subsequently dried under vacuum.
2.3 Electronic circular dichroism and ultraviolet absorption spectroscopy
To obtain the ECD spectra, a J-815 spectrometer (JASCO Corporation, Tokyo, Japan)
purged with nitrogen gas (purity 99.99%, Siad, Czech Republic) during the measurement was
used. The samples of flephedrone, clephedrone and brephedrone were dissolved in
demineralized water (UCT Prague) at a final concentration of 217 mg∙l−1 and norflephedrone,
norclephedrone and norbrephedrone at a concentration of 3.3 g∙l−1 due to high content of
non-absorbing diethylammonium hydrochloride (for details, please see ESI). These solutions
were measured within 185–380 nm at ambient temperature in a quartz cuvette with a path length
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of 1 mm (Hellma, Germany). A 20 nm∙min−1 scanning speed, 8 s response time anDdO1I: 1n0.m103b9/aDn0NdJ05065B
width were applied and three scans of each sample were accumulated and averaged. The
corresponding UV absorption spectra were calculated from the detector HT voltage using the
Spectra Analysis module of the Spectra Manager software (JASCO Corporation, Tokyo,
Japan). To correct the baseline, the spectrum of solvent (demineralized water) measured under
the same conditions was subtracted.
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2.4 Vibrational circular dichroism and infrared absorption spectroscopy
The FTIR IFS 66/S spectrometer equipped with a VCD/IRRAS PMA 37 module
(Bruker, Bremen, Germany), a ZnSe beam splitter (Hinds Instruments, Hillsboro, OR, USA), a
BaF2 polarizer, and an MCT detector (InfraRed Associates, Stuart, FL, USA)31 was used for
the simultaneous acquisition of VCD and IR absorption spectra. The solutions of the individual
samples at a concentration of 150 g∙l−1 in D2O were measured in a BioCell cuvette with CaF2
windows and an optical path length of 27.3 μm (BioTools, Inc., Jupiter, FL, USA) in the spectral
range of 1800–1250 cm−1 with a resolution of 8 cm−1 at ambient temperature. To improve the
signal to noise ratio, the VCD spectra were averaged from 12 blocks, each measured for 20 min
and containing 3680 scans. The IR spectra were obtained by the accumulation of 32 scans. The
baseline of VCD and IR spectra was corrected by subtracting the spectrum of solvent (D2O)
recorded under the same conditions.
2.5 DFT calculations
For correct prediction, the search of the most appropriate position of the chlorine ion
+
related to NH2 group was performed by putting the chlorine ion to different positions and
distances in relation to the molecules32, 33. After the optimization of the chlorine position, the
conformational analysis of (S)-enantiomers of flephedrone, clephedrone and brephedrone
hydrochlorides and their desmethyl derivatives was performed by systematic rotation of their
dihedral angles α1 and α2 (in the case of flephedrone, clephedrone, brephedrone) or only α1 (in
the case of norflephedrone, norclephedrone and norbrephedrone, Fig. 1) using the MCM
software.34 All of those 27 and 9 geometries, respectively, were optimized at the B3LYP/6-31G
level. After that, only two stable conformers with relative energy within 4.2 kcal∙mol−1 of each
substance were selected and further geometry reoptimized at several higher levels of theory
(B3LYP/6-311++G(d,p), B3LYP/ aug-cc-pVTZ, B3PW91/6-311++G(d,p), B3PW91/aug-cc-
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pVDZ, CAM-B3LYP/6-311++G(d,p), CAM-B3LYP/aug-cc-pVTZ, wB97XD/6-311++G
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(d,p), wB97XD/aug-cc-pVTZ, and wB97XD/TZVP; for details, see ESI). Here, in the main
text, we present only the results with the highest agreement with the experimental data
calculated at the B3LYP/6-311++G(d,p) level. The solvent effect of water (ε = 78.3553) was
modelled using a conductor-like polarizable continuum model. The relative abundances of the
stable conformers were calculated using Boltzmann distribution based on the Gibbs free
energies (T = 273 K).
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Fig. 1. Structures of synthetic cathinones (top) and their desmethyl metabolites (bottom) with
labelled dihedral angles α1 and α2 or just α1.
To simulate the ECD and UV spectra, time-dependent (TD) DFT calculations at the
TD-B3LYP/6-311++G(d,p) level during geometry optimization steps were performed and the
lowest 30 singlet states were taken into account. The Gaussian band shape with 28 nm as a half-
height width was applied.
To take into account the hydrogen-deuterium exchange primarily important for the VCD
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were replaced by deuterium isotopes into ND2 in DFT calculations. The VCD and IR bands
were visualised using a Lorentzian function with a half-widths at half maximum of 6 cm−1.
The final Boltzmann-averaged spectra of each method were calculated by summing the
simulated spectra of the individual conformers multiplied by their relative abundances.
Subsequently, those spectra were compared with the experimental spectra using the
CDSpecTech program package28, 35 to gain the scaling factor for simulated spectra and the
similarity index (SimEA, SimECD, SimVA, SimVCD). For perfect agreement of the
experimental and calculated spectra, the similarity index would be 1.0.
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3 Results and discussion
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Generally, the current study presents a complete workflow from the synthesis (seizure)
of a synthetic cathinone down to the full characterization of its 3D structure. The obtained data
are relevant also from a broader point of view, particularly, for the characterization of the
enantiomers of the parent drugs and their major human metabolites in subsequent in vitro and
in vivo studies. Specifically, we have focused on halogen cathinones, which are currently
prevalent in society, however, there is limited information on their 3D structure, toxicity and
other biological properties, especially, for their enantiomers. Therefore, it is assumed that these
studies are being conducted in the near future, and that a methodology for a fast characterization
of the drugs enantiomers is strongly required.
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3.1 Chiral separation
The methodology for the chiral separation of the studied synthetic cathinones and their
metabolites was first developed on an analytical scale. Based on the data available in the
literature for chiral resolution on cathinones using polysaccharide-based columns, we primarily
employed normal phase mode with hexane and heptane as the bulk mobile phase. To ensure
reasonable elution times, EtOH and 2-PrOH were used as polar mobile phase constituents. An
improved peak shape was achieved on the addition of diethylamine (0.1%) as a basic additive
masking the residual silanol units on the surface of the chiral stationary phase. The best results
for the parent drugs were achieved with the mobile phases composed of hexane/2-PrOH (95/5),
RS = 1.55, and heptane/2-PrOH (95/5), RS = 1.11, both with 0.1% DEA (for details, see ESI).
The optimized conditions were adopted for the preparative enantioseparation and further
optimized by reducing the volume of the polar solvent. The performance of the preparative
column slightly differed from the analytical one, and the best results were achieved with a
mobile phase consisting of heptane/2-PrOH (96/4) with 0.1% DEA (Fig. 2). Such low
concentration of alcohol also enabled easy solvent recycling, which significantly reduced the
amount of organic waste produced and the overall separation costs.
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Fig. 2. Preparative chiral separation of (a) flephedrone, (b) clephedrone, and (c) brephedrone.
The intervals of fraction collection are marked by coloured pillars. Conditions: column
Chiralpak IA (250×20 mm, i.d., 5 μm) with the optimum mobile phase; a column temperature
of 15 °C; a flow rate of 10 ml min−1; and a detection wavelength of 254 nm were employed.
The injection volume was 0.5 ml (0.75 ml for brephedrone). The optical purity of the collected
enantiomers was verified in the analytical mode using the Chiral Art Amylose-SA (250×4.6
mm, i.d., 5 μm) column. The analytical purity control chromatograms of (1) flephedrone, (2)
clephedrone, and (3) brephedrone of the first eluting and second eluting enantiomers are shown
in red (upper trace) and blue (lower trace), respectively, in the inset.
A notable improvement of the resolution of enantiomers was observed from flephedrone
to brephedrone, which allowed the injection of larger volumes (a larger amount of
brephedrone). Generally, the yield for all individual enantiomers reached a maximum of 70%
(in the case of flephedrone) of the theoretical amount for the first eluting enantiomer. The loss
was caused by cut-offs of the collected peak areas, which was employed to secure high optical
purity of the individual enantiomers. Additionally, alternatively to various decay pathways, the
relatively rapid cyclization reaction of the cathinone free base may occur, giving rise to a
pyrazine derivative (Fig. 3a),36 thereby leading to a further reduction of the harvested amount
of the target compounds. However, we failed to isolate this cyclization product during the
chromatographic separation. Moreover, it was not possible to prepare the assumed pyrazine
derivative by a targeted synthesis. On the other hand, the cyclization reaction was particularly
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fast for the corresponding nor-derivatives (Fig. 3b), amino function of which is notDhOiI:n1d0.e10r3e9d/Db0NyJ05065B
the methyl substituent.
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X = F, Cl, Br
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CH3
CH3
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Fig. 3. Chemical structures of (a) the assumed cyclization products of the studied parent drugs,
and (b) cyclization product of the nor-derivatives.
The chiral resolution of 4-halogencathinones (nor-derivatives) required a mobile phase
with a higher content of alcohol, due to higher polarity of the substances (ESI, Fig. S1). The
optimum analytical conditions (for hexane/2-PrOH, 80/20/0.1 v/v/v DEA) were also in this case
slightly modified to achieve an efficient base-line separation of the enantiomers of all three
investigated major metabolites. Under these conditions, we were able to achieve chiral
separation of the target nor-derivatives in multi-milligram amounts of each (Fig. 4). As
mentioned above, the products have been separated and isolated as free bases. As such, they
underwent a rapid cyclization reaction, which gave rise to halogenated dihydropyrazines as
major decay products. The increase in the concentration of this unwanted side product was
notable also during the preparative separation documenting their rapid formation. Therefore, to
prevent this reaction, the separated enantiomers were stabilized as hydrochlorides immediately
after separation leading to a mixture of the product and diethylamine hydrochloride. To verify
the nature of the side products, we de novo synthesized and characterized the chloro-substituted
homologue (for details, see ESI).
Although the cyclization reaction of cathinones has been mentioned several times in the
literature, it has only been described scarcely.36 Generally, it is assumed that the cyclization
proceeds via substituted dihydropyrazine, which is immediately oxidized by air oxygen to the
corresponding fully aromatic substituted pyrazine.37 However, our experiments suggest that the
dihydropyrazine species may be rather stable even in the presence of a strong oxidizing agent,
such as hydrogen peroxide and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, as well as in a pure
oxygen atmosphere. These compounds can hardly have been produced during metabolic
transformation of the drugs, however, they may arise during synthesis in clandestine
laboratories. Due to their structure, they may pose an additional risk to the health of the
consumers. Therefore, their biological properties shall be studied in detail.
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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60
Fig. 4. Preparative chiral separation of norflephedrone. The intervals of fraction collection are
marked by coloured pillars. Conditions: column Chiralpak IA (250×20 mm, i.d., 5 μm) with the
optimum mobile phase; a column temperature of 15 °C; a flow rate of 10 mL min−1; and a
detection wavelength of 254 nm were employed. The injection volume was 0.5 ml.
It is interesting to note that the chiral separation of the nor-derivatives was complicated
due to the cyclization of the separated enantiomers (ESI, Fig. S7 and S8). On the other hand,
the harvested enantiomers in the form of hydrochloride salts were stable.
In order to determine the absolute configuration of the separated enantiomers, we used a
combination of DFT calculations and circular dichroism spectra, which has already been
successfully used for this purpose throughout the literature.19, 20, 38
3.2 Conformations
Two stable conformers of flephedrone, brebredrone and clephedrone were revealed in
aqueous solution after conformational search (Fig. 5, Table I). The structural parameters of the
most abundant conformer of each substance (conformer I) were very similar. However, the
dihedral angle α2, determining the orientation of methyl group, of flephedrone II differed from
clephedrone II and brephedrone II. The orientation of this dihedral angle caused the distance
between the oxygen of the carbonyl group and the hydrogen of the amino group to be 2.64 Å
in case of flephedrone II. On the contrary, the rest of all the relevant conformers were stabilized
by the intramolecular hydrogen bond resulting in higher relative abundances of those
conformers than flephedrone II. The length of this bond was 2.19 Å (flephedrone I, clephedrone
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I, brephedrone I) and 2.05 Å (clephedrone II, brephedrone II). It is probable that this was caused
5
6
by the high electronegativity of fluorine.
7
8
9
10
11
12
13
14
15
16
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18
19
20
21
22
23
24
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27
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 5. The most stable conformers of flephedrone, clephedrone and brephedrone
hydrochlorides calculated at the B3LYP/6-311++G(d,p) level.
Table 1. Structural parameters of the most stable conformers of flephedrone, clephedrone and
brephedrone hydrochlorides: dihedral angles α1 and α2, relative Gibbs free energies and relative
abundances. The calculations were performed at the B3LYP/6-311++G(d,p) level.
relative
abundance [%]
conformer
α1 [°]
α2 [°]
ΔG [kJ mol−1]
flephedrone I
flephedrone II
clephedrone I
clephedrone II
brephedrone I
brephedrone II
96
107
96
−79
75
0
4.2
0
99.9
0.1
−78
−142
−79
−142
93.4
6.6
116
97
1.6
0
93.7
6.3
116
1.6
3.3 Electronic circular dichroism and ultraviolet absorption spectroscopy
The ECD measurement of each substance produced a mirror-image ECD spectra for
each two spectra of individual enantiomers (Fig. 6, bottom) and thus confirmed their
enantiomeric character. This method enabled us to distinguish between those three structurally
very similar substances. Despite their very similar spectral shape, all wavelengths were shifted
to higher values when a substituent with a higher atomic weight was presented on the aromatic
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ring. The simulations (Fig. 6, middle) also correctly predicted this phenomenon. ODnOlyI: 1t0h.1e03b9/aDn0NdJ05065B
~300 nm was split into two bands in the experimental spectra while only one band was visible
in the calculated spectra. This band is, according to calculations, associated with the π → π*
electronic transition from the highest occupied molecular orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO), this transition is shown in Fig. 7. The ECD spectra of
individual conformers had similar spectral shape in the case of flephedrone, bigger differences
could be seen in spectra of clephedrone and brephedrone. The experimental and simulated
spectra were considered to be in good agreement with the similarity index of 0.76, 0.82 and
0.88 for flephedrone, clephedrone and brephedrone, respectively.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
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19
20
21
22
23
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 6. Comparison of ECD spectra of flephedrone (left), clephedrone (middle) and brephedrone
(right) hydrochlorides in H2O solution: simulated spectra of the individual conformers at the
TD-B3LYP/6-311++G(d,p) level (top), their population weighted spectra (middle) and the
experimental spectra (bottom).
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6
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53
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59
60
Fig. 7. Electronic transition from the highest occupied molecular orbital (HOMO, bottom) to
the lowest unoccupied molecular orbital (LUMO, top) of the most abundant conformer of
flephedrone, clephedrone and brephedrone hydrochlorides simulated at the TD-B3LYP/6-
311++G(d,p) level.
To distinguish flephedrone, clephedrone and brephedrone by the UV absorption
experimental spectra (Fig. 8, bottom), the band ~260 nm which was shifted again (as seen in
ECD spectra) depending on the different substituent used. The relative intensities of the band
shoulders ~200 nm were also different, but this change was slight. These shoulders were not
predicted by the TD-DFT calculations, only one broad band ~190 nm was present in all
simulated spectra. However, the final similarity index was 0.91, 0.92 and 0.88 for flephedrone,
clephedrone and brephedrone, respectively.
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6
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 8. Comparison of UV absorption spectra of flephedrone (left), clephedrone (middle) and
brephedrone (right) hydrochlorides in H2O solution: simulated spectra of the individual
conformers at the TD-B3LYP/6-311++G(d,p) level (top), their population weighted spectra
(middle) and the experimental spectra (bottom).
3.4 Vibrational circular dichroism and infrared absorption spectroscopy
First, the mirror image of the experimental VCD spectra (Fig. 9, bottom) of the
individual samples of each substance confirmed the enantiomers were obtained. Subsequently,
the correct assignment of the absolute configuration of enantiomers was confirmed according
to the comparison of the experimental and population weighted VCD spectra (Fig. 9, bottom
and middle). The similarity index reached very convincing values: 0.90, 0.91, 0.86 for
flephedrone, clephedrone, brephedrone, respectively. According to another comparison method
for VCD experimental and calculated spectra39,40, the similarity index reached also very
satisfying values of 0.75, 0.76 and 0.64 for flephedrone, clephedrone, brephedrone,
respectively. The VCD spectra of the individual enantiomers (Fig. 9, top) showed a similar
shape except for the band ~1470 cm−1 reflecting the deformation vibrations of the –CH3 group
(band 4 for flephedrone, clephedrone and brephedrone) and band ~1680 cm−1 in the spectra of
clephedrone and brephedrone reflecting the stretching vibrations of the carbonyl group (band
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1). The different number of bands, their relative intensities and the band positionDsO, Ie: 1s0p.1e0c39i/aDl0lNyJ05065B
below 1500 cm−1, enabled us to distinguish those three structurally similar compounds due to
the high sensitivity of the VCD spectroscopy when compared to ECD.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 9. Comparison of VCD spectra of flephedrone (left), clephedrone (middle) and
brephedrone (right) hydrochlorides in D2O solution: simulated spectra of the individual
conformers at the B3LYP/6-311++G(d,p) level (top), their population weighted spectra
(middle) and experimental spectra with the spectra of noise (bottom). VCD band numbering
corresponds to IR band numbering. Bands 6’ come from the same normal mode as bands 6.
After the spectra comparison, the similarity index of the IR absorption experimental and
simulated spectra (Fig. 10, bottom and middle, Table II–IV) reached 0.84, 0.78 and 0.77 for
flephedrone, clephedrone and brephedrone, respectively. The simulated spectra of the
individual conformers of each substance (Fig. 10, top) had a similar shape due to the lower
sensitivity of IR absorption spectroscopy to the 3D structure. Flephedrone could be relatively
easily distinguished from the two other substances in the spectral region of 1600–1500 cm−1
where only two bands could be found, whereas in the spectra of clephedrone and brephedrone
two shoulders of them were also visible. The other region where flephedrone could be
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distinguished is ~1390 cm−1 with a weak band. Clephedrone and brephedrone could not be
5
6
7
convincingly distinguished from each other in the IR absorption spectral region of 1800–1250
cm−1.
8
9
10
11
12
13
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18
19
20
21
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 10. Comparison of IR absorption spectra of flephedrone (left), clephedrone (middle) and
brephedrone (right) hydrochlorides in D2O solution: simulated spectra of the individual
conformers at the B3LYP/6-311++G(d,p) level (top), their population weighted spectra
(middle) and the experimental spectra (bottom).
Table 2. Experimental and simulated [B3LYP/6-311++G(d,p)] frequencies and the assignment
of the vibrational modes for IR absorption spectrum of flephedrone hydrochloride.
band
number
experimental
simulated spectrum
[cm−1]
vibrational mode
spectrum [cm−1]
1
2
3
4
5
6
7
1688
1601
1512
1460
1418
1394
1306
1685
1604
1513
1472
1421
1399
1303
ν (C=O)
ν (C=C), Ar
δ (N–H)
δ (C–H), –CH3
δ (C–H), Ar, –CH
δ (C–H)
δ (C–H), –CH–CH3
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δ (C–H), ν (C–C) =C–C=O
8
1252
1252
5
6
7
Table 3. Experimental and simulated [B3LYP/6-311++G(d,p)] frequencies and the assignment
of the vibrational modes for IR absorption spectrum of clephedrone hydrochloride.
8
9
band
number
experimental
simulated spectrum
[cm−1]
vibrational mode
spectrum [cm−1]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
3
4
5
6
7
8
1689
1591
1574
1491
1460
1408
1302
1252
1686
1597
1579
1497
1471
1411
1304
1252
ν (C=O)
δ (N–H), ν (C=C), Ar
ν (C=C), Ar
δ (N–H)
δ (C–H), –CH3
δ (C–H), ν (C=C), Ar
δ (C–H), –CH–CH3
δ (C–H), ν (C–C) =C–C=O
Table 4. Experimental and simulated [B3LYP/6-311++G(d,p)] frequencies and the assignment
of the vibrational modes for IR absorption spectrum of brephedrone hydrochloride.
band
number
experimental
simulated spectrum
[cm−1]
vibrational mode
spectrum [cm−1]
1
2
3
4
5
6
7
8
1689
1587
1570
1488
1460
1404
1303
1252
1687
1593
1576
1495
1472
1408
1307
1252
ν (C=O)
δ (N–H)
ν (C=C), Ar
δ (N–H)
δ (C–H), –CH3
δ (C=C), Ar
δ (C–H), –CH–CH3
δ (C–H), ν (C–C) =C–C=O
3.5 Electronic circular dichroism and ultraviolet absorption spectroscopy of desmethyl
derivatives
Since the desmethyl derivatives are assumed to be the major metabolites of the studied
synthetic cathinones21, it is important to obtain information on their 3D-structure. The
enantiomeric character of the individual enantiomers of the studied desmethyl derivatives
(norflephedrone, norclephedrone and norbrephedrone) was confirmed through their mirror-
image experimental ECD spectra (Fig. 11, bottom). Their simulated population weighted ECD
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spectra (Fig. 11, middle) evinced good agreement of 0.75, 0.84 and 0.74 for norDflOeIp: 1h0.e10d3r9o/Dn0eN,J05065B
norclephedrone and norbrephedrone, respectively. In comparison to the experimental ECD
spectra of flephedrone, clephedrone and brephedrone (Fig. 3), the experimental bands were of
the same wavelength or shifted by 1–2 nm.
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11
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41
42
43
44
45
46
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48
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50
51
52
53
54
55
56
57
58
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60
Fig. 11. Comparison of ECD spectra of norflephedrone (left), norclephedrone (middle) and
norbrephedrone (right) hydrochlorides in H2O solution: simulated spectra of the individual
conformers at the TD-B3LYP/6-311++G(d,p) level (top), their population weighted spectra
(middle) and the experimental spectra (bottom).
The analyses of the desmethyl derivatives performed by UV absorption spectroscopy
and related calculations were presented in ESI (Fig. S9). However, for lack of those substances,
we were not able to measure the VCD and IR absorption spectra.
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4 Conclusion
In this study, we have focused on the chiral separation of three prevalent new
psychoactive substances from the family of synthetic cathinones (flephedrone, clephedrone,
brephedrone). We have elaborated the method for their chiral separation on the multi-milligram
scale using a polysaccharide-based chiral stationary phase. Successful enantioseparation has
been achieved also for de novo synthesized assumed major metabolites of the studied drugs
(norflephedrone, norclephedrone, norbrephedrone). Subsequently, we have successfully
combined chiroptical, vibrational and electronic spectroscopies with ab initio calculations to
obtain the 3D structures of the parent drugs in aqueous solution. Using these powerful methods,
two stable conformers of each substance were identified and their relative abundances
calculated. Very good agreement between the experimental and simulated spectra enabled us
to assign the absolute configuration of the individual enantiomers and revealed the structures.
ECD spectra (theoretical and experimental) have provided sufficiently strong information to
determine the absolute configuration of the corresponding nor-derivatives. Furthermore, our
experiments showed that the chiroptical methods used are suitable for distinguishing between
structurally very similar compounds. The obtained knowledge can broaden the understanding
of the processes of NPS metabolism in the human organism.
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7
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60
Conflict of interest
The authors declare that they have no conflict of interest.
Funding
This work was supported by the Ministry of the Interior of the Czech Republic
[MV0/VI20172020056]. Partial support (infrastructure and instrumentation) was provided by
the Operational Program Prague
–
Competitiveness [CZ.2.16/3.1.00/24503 and
CZ.2.16/3.1.00/21537]; the National Program of Sustainability [NPU MSMT – LO1601;
43760/2015]; and the grant of Specific university research [A2_FCHI_2020_016 and
A2_FCHT_2020_043].
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New Journal of Chemistry
Page 22 of 22
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View Article Online
DOI: 10.1039/D0NJ05065B
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We present chiral semipreparative methodology and a comprehensive spectroscopic structural study
of three synthetic cathinones and their expected desmethyl metabolites.