Structure determination of butylone as a new psychoactive substance using chiroptical and vibrational spectroscopies

Article abstract of DOI:10.1002/chir.22825

Recently, there has been a worldwide substantial increase in the consumption of new psychoactive substances (NPS), compounds that mimic the structure of illicit drugs, such as amphetamines or ecstasy. The producers try to avoid the law by a slight modification of illicit structures, thereby developing dozens of temporarily legal NPS every year. The current trends in the detection and monitoring of such substances demand a fast and reliable analysis. Molecular spectroscopy represents a highly effective tool for the identification of NPS and chiroptical methods can provide further information on their 3D structure, which is the key for the determination of their biological activity. We present the first systematic study of NPS, specifically butylone, combining chiroptical and vibrational spectroscopies with ab initio calculations. According to density functional theory calculations, 6 stable lowest energy conformers of butylone were found and their molecular structure was described. For each conformer, the relative abundance based on the Boltzmann distribution was estimated, their population weighted spectra predicted and compared to the experimental results. Very good agreement between the experimental and the simulated spectra was achieved, which allowed not only the assignment of the absolute configuration, but also a precise description of the molecular structure.

Full text of DOI:10.1002/chir.22825

Received: 25 October 2017
DOI: 10.1002/chir.22825
Revised: 2 January 2018
Accepted: 3 January 2018
S P E C I A L I S S U E A R T I C L E
Structure determination of butylone as a new psychoactive
substance using chiroptical and vibrational spectroscopies
Dita Spálovská1
Lucie Habartová1
| František Králík1
| Martin Kuchař2,4
| Michal Kohout3
| Vladimír Setnička1
| Bronislav Jurásek2,4
|
1 Department of Analytical Chemistry,
University of Chemistry and Technology,
Prague 6, Czech Republic
2 Forensic Laboratory of Biologically
Active Substances, University of
Chemistry and Technology, Prague 6,
Czech Republic
3 Department of Organic Chemistry,
University of Chemistry and Technology,
Prague 6, Czech Republic
4 Department of Chemistry of Natural
Compounds, University of Chemistry and
Technology, Prague 6, Czech Republic
Abstract
Recently, there has been a worldwide substantial increase in the consumption
of new psychoactive substances (NPS), compounds that mimic the structure
of illicit drugs, such as amphetamines or ecstasy. The producers try to avoid
the law by a slight modification of illicit structures, thereby developing dozens
of temporarily legal NPS every year. The current trends in the detection and
monitoring of such substances demand a fast and reliable analysis. Molecular
spectroscopy represents a highly effective tool for the identification of NPS
and chiroptical methods can provide further information on their 3D structure,
which is the key for the determination of their biological activity. We present
the first systematic study of NPS, specifically butylone, combining chiroptical
and vibrational spectroscopies with ab initio calculations. According to density
functional theory calculations, 6 stable lowest energy conformers of butylone
were found and their molecular structure was described. For each conformer,
the relative abundance based on the Boltzmann distribution was estimated,
their population weighted spectra predicted and compared to the experimental
results. Very good agreement between the experimental and the simulated
spectra was achieved, which allowed not only the assignment of the absolute
configuration, but also a precise description of the molecular structure.
Correspondence
Vladimír Setnička, Department of
Analytical Chemistry, University of
Chemistry and Technology, Technická 5,
16628 Prague 6 (Czech Republic).
Email: vladimir.setnicka@vscht.cz
Funding information
Ministry of the Interior of the Czech
Republic, Grant/Award Number: MV0/
VI20172020056; Operational Program
Prague—Competitiveness, Grant/Award
Number: CZ.2.16/3.1.00/24503 and
CZ.2.16/3.1.00/21537; National Program of
Sustainability, Grant/Award Numbers:
NPU MSMT—LO1601 and 43760/2015;
Specific University Research, Grant/
Award Number: MSMT No. 20‐SVV/2017
and 20‐SVV/2018
KEYWORDS
ab initio calculations, butylone, chiral separation, chiroptical spectroscopy, molecular spectroscopy,
new psychoactive substances, preparative enantioseparation, synthetic cathinone
1 | INTRODUCTION
of new substances occurring during the year) and conse-
quent availability (Internet sales), there is a significant
New psychoactive substances (NPS) are often being sold
as a legal alternative to already prohibited psychoactive
drugs. NPS exhibit analogous effects, which are achieved
by a slight modification of the chemical structure of illicit
drugs while not altering the pharmacophore.1 Because of
the complicated legislative regulation (large increase
increase in the prevalence of these substances within the
population.2
NPS are pharmacologically unidentified xenobiotics
with unknown effects and toxicity, which entail consider-
able health risks for the consumers.3,4 Unfortunately, the
examination of their biological properties and the
Chirality. 2018;1–12.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
SPÁLOVSKÁ ET AL.
assessment of the health hazards after their use is not pos-
sible in the short term because in‐depth pharmacological,
toxicological, and behavioral studies are expensive and
time‐consuming. Since NPS pose a serious health and
sociopathological risk for the society, great efforts are
being invested into the development of sensitive analyti-
cal detection methods. Ideally, these methods should
ensure a reliable identification (or even quantification)
of these substances in a pure form as well as in various
matrices.5
Raman spectroscopy and high‐performance liquid
chromatography with mass detection (HPLC‐MS) are the
most commonly used techniques for the identification of
pure substances.6 HPLC‐MS is further used for the detec-
tion of NPS in biological samples (eg, urine, serum),
which is absolutely crucial in medical and toxicological
practice.7
The issues of NPS monitoring associated with the
detection, health risk assessment, and legislative regula-
tions are further complicated because of the increasing
number of newly seized substances on the black market
per year, and the lack or often complete absence of analyt-
ical standards.
Synthetic cathinones, also called “bath salts,” are
among the most commonly seized NPS in Europe.1 This
group of substances gained their name from the
cathinone structure present within the active substance
of Catha edulis, which is typically cultivated in the Horn
of Africa and on the Arabian Peninsula.8 Cathinones have
an amphetamine‐like pharmacological profile, which
might be caused by an interaction with dopamine and
5‐hydroxytryptamine (5‐HT) receptors and transporters.9
Cathinone overdose may lead to psychosis, which is
attributed to the interaction with the 5‐HT2A receptor.10
Butylone (Figure 1) is a synthetic cathinone that
emerged on the European drug market as a new designer
drug in 2008 (Figure 2). Like its natural analogue,
of pure enantiomers (eg, stereoselective synthesis, chiral
separation) and methods that can be used to differentiate
between the enantiomers of a particular drug.
Chiroptical spectroscopy, specifically Raman optical
activity, electronic circular dichroism (ECD), and vibra-
tional circular dichroism (VCD), combined with quan-
tum‐chemical calculations are an efficient tool for the
determination of the absolute configuration of separated
enantiomers and their molecular stereochemistry.15-24
The ab initio calculations allow a detailed interpretation
of the experimental spectra of chiral molecules in solution.
It has been demonstrated that the application of several
independent chiroptical methods for the determination of
the 3D structure of a chiral substance significantly
increases the reliability of the obtained results.25
In this study, we present a chiral chromatographic
separation protocol suitable for the enantiomer purity
analysis. Our method was used for the separation of
several milligrams of butylone. Furthermore, chiroptical
and vibrational spectroscopic analyses combined
with density functional theory calculations at the
B3LYP/6‐311++G(d,p) level are used to elucidate the
absolute configuration and to determine the 3D structure
of stable butylone conformers.
2 | MATERIALS AND METHODS
2.1 | Chemicals, reagents
Racemic butylone hydrochloride was purchased as stan-
dard (98%) from Alfarma, Czech Republic. The solvents
for analytical and preparative chromatography were of
HPLC grade and obtained from LachNer, Czech Republic.
Deuterium oxide (D2O; 99.9% D) for the dissolution of
butylone was supplied by ISOSAR GmbH, Germany.
2.2 | Enantioseparation
butylone
exhibits
psychostimulant
effects
(eg,
hyperlocomotion) and causes the increase of extracellular
dopamine concentration.9 Furthermore, it acts as a low
potency inhibitor of the dopamine (DAT), norepinephrine
(NET), and serotonine (SERT) transporters.9
Both the analytical and the preparative enantioseparation
of butylone enantiomers was performed on the Waters
AutoPurification System (Waters, United States) equipped
with a UV‐Vis detector (detection wavelength of 254 nm).
The analytical separation was performed using a chiral
Most studies11-13 published thus far have been focused
on racemic cathinones, and stereo‐specific drug interac-
tions have been demonstrated for amphetamines.14 For
studies on cathinones, which might reveal significant
pharmacological differences between particular enantio-
mers, it is essential to develop methods for the production
polysaccharide
column
ChiralArt
Amylose‐SA
250 × 4.6 mm, a particle diameter of 5 μm, with an analyte
concentration of 1 mg mL−1 and a flow rate of 1 mL min−1
at ambient temperature. For the preparative chromatogra-
phy, an analogous polysaccharide column available in our
laboratory—Chiralpak IA 250 × 20 mm with a particle
diameter of 5 μm—was used with the analyte concentra-
tion of 5 mg mL−1 and a flow rate of 15 mL min−1 at ambi-
ent temperature. For analytical chromatography
screening, commercially available butylone hydrochloride
FIGURE 1 Structures of butylone enantiomers

SPÁLOVSKÁ ET AL.
3
FIGURE 2 Butylone occurrence in
Europe
was dissolved in the mobile phase containing 0.1% of
diethylamine. The sample for preparative chromatography
(5 mg) was dissolved in a mixture of 1% diethylamine in
propan‐2‐ol (0.45 mL), and a total volume of 1 mL was
achieved by the addition of heptane. In both cases, the
addition of diethylamine ensured the transformation of
butylone hydrochloride to butylone free base.
under identical experimental conditions. The correspond-
ing UV absorption was calculated from the detector HT
voltage using the Spectra Analysis module of the Spectra
Manager software (Jasco, Japan).
2.4 | Vibrational circular dichroism
The VCD and IR absorption spectra were recorded on the
FTIR IFS 66/S spectrometer equipped with a VCD/IRRAS
PMA 37 module (Bruker, Germany), a ZnSe beam splitter
(Hinds Instrument, United States), a BaF2 polarizer, and
an MCT detector (InfraRed Associates, United States).26
The spectra were measured at ambient temperature in a
spectral region of 1750 to 1250 cm−1 with a resolution of
8 cm−1. The solution of (R)‐butylone hydrochloride in
D2O (100 mg mL−1) was placed into a BioCell cuvette
with CaF2 windows (BioTools, Inc, United States) and
an optical pathlength of 27.3 μm. The VCD spectra were
averaged from 9‐12 blocks, each containing 2260 scans.
The baseline was corrected by the subtraction of solvent
spectra measured under identical experimental
2.3 | Electronic circular dichroism
The ECD spectra of the aqueous solution of (R)‐butylone
hydrochloride at a concentration of 15 mg L−1 were
recorded using the J‐815 spectrometer (Jasco, Japan). In
a spectral range of 380 to 185 nm, the prepared solution
was measured in a quartz cell (Hellma, Germany) with a
fixed pathlength of 1 cm. The scanning speed was set to
20 nm min−1, the response time to 8 seconds, and the
band width to 1 nm. All measurements were performed
at ambient temperature. The final spectra were averaged
from 3 accumulations. The baseline was corrected by
subtracting the spectra of demineralized water obtained

4
SPÁLOVSKÁ ET AL.
conditions. The final VCD spectrum was obtained by the
arithmetic subtraction of the VCD spectra of (R)‐butylone
and (S)‐butylone and division by a factor of 2.
All simulated spectra were calculated at the B3LYP/6‐
311++G(d,p) level with the conductor‐like polarizable
continuum model. The deuteration of amine hydrogens
and its effect on the resulting VCD spectra was consid-
ered. To visualize the simulated VCD, IR, and Raman
spectra, Lorentzian broadening with a 10‐cm−1 band-
width at half‐maximum was used. For ECD and UV
absorption, the 30 lowest energy transitions of each con-
former were calculated. In this case, Gaussian broadening
with 12‐nm bandwidth at half‐maximum was applied for
the visualization of the calculated spectra.
2.5 | Raman spectroscopy
The Raman spectra were recorded on the DXR
SmartRaman spectrometer (Thermo Scientific, United
States) equipped with an excitation wavelength of 532 nm
and a diffraction grid comprised of 900 lines per mm. The
samples (40 μL) of (R)‐butylone hydrochloride in
demineralized water at a concentration of 100 mg mL−1
were placed into a 3 × 4 × 10 mm optical cell with an anti-
reflective coating (BioTools, Inc, United States) and
3 | RESULTS AND DISCUSSION
3.1 | Enantioseparation of butylone
measured within a spectral region of 200 to 2500 cm−1
.
To acquire reliable spectra, the laser power on the sample
and the slit width were set to 10 mW and 25 μm, respec-
tively. Background fluorescence was suppressed by
photobleaching at 10 mW for 24 hours. To correct the raw
Cathinones can generally be enantioseparated using vari-
ous methods including liquid chromatography, gas chro-
matography, supercritical fluid chromatography, and
capillary electrophoresis.31-34 Since the substance is avail-
able as hydrochloride, the polar organic mode arrange-
ment with ethanol as the bulk mobile phase was
assumed to provide the best results. Although the
enantioseparation of a series of cathinones in the polar
organic mode has been reported recently,35 butylone was
not included in the study. Therefore, screening of a
mobile phase for the separation of butylone enantiomers
was performed. Under polar organic mode conditions,
the enantiomers were not separated. This indicated that
the interaction of the amino group of butylone with the
selected polysaccharide chiral stationary phase is crucial
spectra for residual baseline distortion, we modified27
a
procedure described in the literature.28 The spectra were
highly smoothed to create a virtual baseline, which was
subsequently subtracted from the raw spectra. No addi-
tional smoothing or processing was performed.
2.6 | Ab initio calculations
Selected dihedral angles were systematically varied to
obtain the starting geometries of (R)‐butylone hydrochlo-
ride. Geometry optimization of 6 final conformers was
performed at various levels of theory (B3LYP/6‐311+
+G(d,p), B3PW91/aug‐cc‐pVDZ, B3PW91/6‐311++G(d,
p), CAM‐B3LYP/6‐311++G(d,p), CAM‐B3LYP/aug‐cc‐
pVDZ, wB97XD/6‐311++G(d,p), wB97XD/aug‐cc‐pVDZ,
wB97XD/TZVP, Table S1) with the Gaussian 09 program
package.29 Only the results providing the best level of
agreement with the experimental spectra will be
described further in this work. The results of other levels
of theory (also listed in Table S1) did not lead to a con-
vincing improvement in accuracy.
The solvation effect of D2O was considered by
implementing the conductor‐like polarizable continuum
model. The hydrogen‐deuterium exchange of the amine
group and its effect on the simulated spectra was also con-
sidered. The population weighted average VCD, ECD, IR,
UV, and Raman spectra of the low‐energy conformers
were calculated using Boltzmann weights based on Gibbs
free energies at a temperature of 298 K. The experimental
and calculated spectra were compared using the
CDSpecTech program, which employs the enantiomeric
similarity index algorithm to determine the optimal
scaling factor.30
FIGURE 3 Preparative enantioseparation of butylone with the
marked boundaries for the collection of butylone 1 and butylone 2.
The insets show the analytical control of the optical purity of the
collected enantiomers: butylone 1 (A) and butylone 2 (B)

SPÁLOVSKÁ ET AL.
5
for chiral recognition. Therefore, we transformed the ana-
lyte to a free base by adding diethylamine into the sample
and the mobile phase. The added base not only secures
the hydrochloride‐to‐free‐base transformation but also
masks the residual hydroxyl units of the silica solid sup-
port, thus improving the peak shape of eluting enantio-
mers. The butylone free base was analyzed using several
mobile phases with various contents of hexane (heptane)
and propan‐2‐ol (ethanol). The best results were achieved
with a mobile phase composed of hexane/propan‐2‐ol
(95/5, v/v) with 0.1% (v) diethylamine, which yielded the
highest resolution of butylone enantiomers in analytical
as well as in preparative chromatography (Figure 3).
The limits for peak collection were set to ensure high
purity of the enantiomers. The collected fractions were
evaporated separately, and the products were dried under
vacuum. To assure the stability of the separated enantio-
mers and to avoid a cyclization reaction occurring for
the cathinone free base,36 the separated substances were
transformed to hydrochlorides using an etheric solution
of hydrogen chloride and subsequently analyzed in the
analytical mode. The first eluting enantiomer (butylone
1) was found to be optically pure with the enantiomeric
excess of >99.5%. The purity of the second eluting enan-
tiomer (butylone 2) was 98.7%, which was still acceptable
for the following analyses.
3.2 | Conformational analysis
For the theoretical simulations, the (R)‐enantiomer of
butylone hydrochloride was selected. Conformational
options of this molecule are defined by 4 dihedral angles
α1, α2, α3, and α4 (Figure 4). Using the MCM software37
for the conformational search, α1 was varied with a step
of 180°; α2, α3, and α4 were modified with a step of 120°.
As a result, 54 starting conformers were obtained and sub-
sequently optimized at the B3LYP/6‐31+G(d) level. The
conformational analysis revealed all relevant lowest
energy stable conformers considering their Gibbs free
FIGURE 4 Structure of butylone hydrochloride with labeled
dihedral angles α1, α2, α3, α4
FIGURE 5 Structures of 6 lowest energy conformers of (R)‐butylone hydrochloride calculated at the B3LYP/6‐311++G(d,p) level; the
structures are displayed with the identical orientation of the aromatic ring

6
SPÁLOVSKÁ ET AL.
15
10
5
energies. Most conformers either converged to the same
geometry or their population was too low, and therefore,
only 6 stable conformers (Figure 5) were subjected to fur-
ther analysis. The geometry optimization for these con-
formers was performed at a more refined level of theory,
ie, B3LYP/6‐311++G(d,p). The Boltzmann distribution
was used to calculate the relative abundances of the con-
formers (Table 1). The α1 dihedral angle, which defines
the orientation of the carbonyl group with respect to the
aromatic ring, preferably adopts 2 opposite positions.
Conformers I, II, and VI exhibit α1 values of approxi-
mately −7°, whereas α1 reaches −170° for conformers
III, IV, and V (Table 1). The second dihedral angle (α2)
describes the orientation of the alkyl moiety and occupies
preferably 2 orientations. The major difference between
the studied conformers is the orientation of the ethyl moi-
ety, which is characterized by α4. α3 showing the orienta-
tion of the methylamino group does not reflect any
significant difference between the conformers.
0
(R)-I
-5
-10
-15
4
(R)-II
(R)-III
(R)-IV
(R)-V
(R)-VI
B3LYP/6-311++G(d,p)
185
2
335
238
0
-2
-4
-6
4
202
299
(R)-butylone
274
experimental
butylone 1
186
330
235
2
0
3.3 | Electronic circular dichroism
302
-2
-4
-6
205
The mirror‐image ECD spectra of the first and second
eluting compound (Figure 6) clearly document their
enantiomeric character. According to the comparison
of the experimental and simulated spectra, the absolute
configuration of butylone 1 and 2 was determined as
the (S)‐ and (R)‐enantiomer, respectively. To assign the
bands in the experimental and simulated spectra
correctly, the resulting spectrum was scaled by a factor
of 0.99 (based on the similarity plot presented in
Figure 7A) and the similarity factor reached 0.804.
Eventually, the absolute configuration was confirmed
by a detailed analysis of not only the ECD, but also
the VCD spectra (vide infra).
butylone 2
300
278
200
250
350
wavelength [nm]
FIGURE
6
Calculated ECD spectra of
6
(R)‐butylone
hydrochloride conformers (top), their average spectrum (middle),
and experimental (bottom) ECD spectra of butylone 1 and butylone 2
observed: positive bands at 186, 235, and 330 nm, and
negative bands at 205, 278, and 302 nm. The bands at
~186, 205, and 235 nm reflect the combination of
π → π*, n → π*, and n → σ* electronic transitions. The
band at 330 nm is caused mainly by the electronic transi-
tion from the highest occupied to the lowest unoccupied
molecular orbitals (HOMO → LUMO, Figure 8). The
same HOMO → LUMO transition significantly contrib-
utes to the band arising at 278 nm.10 Three positive and
The simulated ECD spectra of the individual con-
formers of (R)‐butylone hydrochloride, the final popula-
tion weighted spectrum and the experimental spectra of
both butylone hydrochloride enantiomers are shown in
Figure 6. In the experimental spectra, 6 bands were
TABLE 1 Six stable conformers of (R)‐butylone hydrochloride, their relative Gibbs free energies, Boltzmann populations, and values of
dihedral angles α1, α2, α3, and α4 calculated at B3LYP/6‐311++G** level
Conformer
ΔG, kJ mol−1
Population, %
α1, °
−8
α2, °
α3, °
α4, °
57
I
0
37
−101
83
II
1.1
1.4
3.3
4.3
8.6
24
21
10
7
−7
−170
−172
−168
−7
−83
−101
−101
−85
73
83
89
75
85
−68
58
III
IV
V
−165
−67
−166
VI
1
−98

SPÁLOVSKÁ ET AL.
7
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
1.0
0.8
0.6
0.4
0.2
0.0
(A)
(B)
0.8 0.9 1.0 1.1 1.2 1.3
wavelength scale factor
0.8 0.9 1.0 1.1 1.2 1.3
wavelength scale factor
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
(C)
(D)
0.96
0.98
1.00
0.96
0.98
1.00
wavenumber scale factor
wavenumber scale factor
1.0
(E)
0.8
0.6
0.4
0.2
0.0
FIGURE 7 Similarity plots for the
calculated (B3LYP/6‐311++G(d,p)) and
experimental ECD (A), UV (B), IR (C),
VCD (D), and Raman (E) spectra of (R)‐
butylone hydrochloride. The scale factors
reveal the maximum similarity available
among the experimental and calculated
spectra
0.96 0.98 1.00 1.02 1.04
wavenumber scale factor
3 negative bands dominated the simulated population
weighted spectra as well. The positive bands were located
at 185, 238, and 335 nm with the negative bands at 202
and 274 nm and a shoulder at 299 nm.
with the similarity factor of 0.787, good agreement in the
band positions between the experimental and the simu-
lated spectra was found.
The simulated UV absorption spectra of individual
conformers, the final population weighted spectrum, and
the comparison with the experimental spectra of both
enantiomers are shown in Figure 9. The experimental
spectra showed at least 4 distinct absorption bands at
195 (structured), 235, 282 and 321 nm. Similar values
were obtained for the predicted absorption bands of the
population weighted spectrum (193, 235, 271, and
327 nm). Since the band positions in the calculated spec-
trum slightly differed from those in the experiment, the
frequencies in the simulated spectra were scaled by 0.98.
According to the similarity overlap plot (Figure 7b) along
3.4 | Vibrational spectroscopy
The calculated IR absorption spectra of individual con-
formers, the final population weighted spectrum, and the
experimental spectrum of the (R)‐butylone enantiomer
are shown in Figure 10. Nine IR absorption bands are pres-
ent in the region of 1750 to 1250 cm−1. These bands were
also correctly predicted by the calculations. To accurately
assign band positions, the frequencies in the simulated
spectra were scaled by 0.983 with similarity factor reaching
0.923 (similarity overlap plot shown in Figure 7C).

8
SPÁLOVSKÁ ET AL.
1.0
0.8
0.6
0.4
0.2
(R)-I
(R)-II
(R)-III
(R)-IV
(R)-V
(R)-VI
0.0
0.8
B3LYP/6-311++G(d,p)
193
327
(R)-butylone
235
0.6
0.4
0.2
271
0.0
0.8
235
235
experimental
experiment
195
195
(S)-butylone
(S)-butylone
(R)-butylone
(R)-butylone
FIGURE 8 Highest occupied molecular orbital (bottom) and
lowest unoccupied molecular orbital (top) calculated at the B3LYP/
6‐311++G(d,p) level for conformer I of (R)‐butylone hydrochloride
0.6
0.4
0.2
0.0
332211
282
282
The calculations enabled us to interpret individual
vibrational modes (Table 2). We achieved very good
agreement between the simulated and the experimental
spectra in band positions as well as relative intensities.
The absorption band located at 1673 cm−1 in the experi-
mental spectrum and at 1670 cm−1 in the simulated spec-
trum is caused by the stretching vibration of C═O. The
presence of the aromatic ring is indicated by the C═C
stretch at 1618 and 1630 cm−1 in the experimental and
simulated spectra, respectively. The band related to the
NH2 stretching vibration is observed at 1606 cm−1 (exper-
imental spectrum) and 1609 cm−1 (simulated spectrum).
The simulated VCD spectra of individual conformers,
the final population weighted spectrum, and the corrected
experimental spectrum of (R)‐butylone hydrochloride
with the VCD noise spectrum are shown in Figure 11.
The spectral region of the experimental spectrum is 1750
to 1263 cm−1 because of a high level of the noise that is
caused by high absorption at lower frequencies. The band
positions in the simulated spectra were scaled by 0.9785
(Figure 7D), and the similarity factor reached 0.648, when
the reliable spectral region of the experimental spectrum
was set to 1650 to 1263 cm−1. According to Covington
and Polavarapu,38 this kind of result achieved using
CDSpecTech indicates a reliable assignment of the abso-
lute configuration and structural properties.
200
250
300
350
wavelength [nm]
FIGURE 9 Calculated UV absorption spectra of 6 (R)‐butylone
hydrochloride conformers (top), their average spectrum (middle),
and the experimental spectrum of (R)‐ and (S)‐butylone
hydrochloride (bottom)
in the experiment, probably because of mode splitting.
In addition, the carbonyl stretching modes are very sensi-
tive to solvation effects.39 Therefore, this part of the spec-
tral region was not considered reliable for determining
the spectra similarity. In the spectral region measured,
we observed 9 significant VCD bands, all of which have
corresponding bands in the simulated population
weighted spectrum. The spectra are very similar to each
other in terms of the relative intensities, positions, and
sign of the bands (Table 3).
Additionally, simulated Raman spectra of individual
conformers, the final population weighted spectrum, and
the experimental spectrum of the (R)‐enantiomer are
shown in Figure 12. The experimental spectrum contained
a number of significant well‐resolved bands being in very
good agreement with those predicted within the popula-
tion weighted spectrum. A scaling factor of 0.982 was used
to achieve a similarity overlap of 0.746 (Figure 7E).
On the basis of the abovementioned experimental
and computational approaches, we confirmed the
The negative bands of the carbonyl stretching vibra-
tion (1 in Figure 11) are well separated in the simulated
spectrum, whereas a broad negative band was observed

SPÁLOVSKÁ ET AL.
9
1.0
0.8
0.6
0.4
0.2
0.0
0.8
-6
-6
20x10
10x10
(R)-I
(R)-II
(R)-III
(R)-IV
(R)-V
(R)-VI
0
-6
-10x10
-20x10
-6
-6
-6
-6
6x10
4x10
2x10
B3LYP/6-311++G(d,p)
9
0
0.6
-6
-2x10
-4x10
-6x10
-8x10
6
-6
-6
-6
0.4
1
3
5
0.2
2
4
8
7
-6
-10x10
15x10
-6
0.0
0.6
experimental
9
-6
-6
10x10
5x10
0.5
0.4
6
0
0.3
1
-6
-5x10
5
4
0.2
0.1
3
-6
8
-10x10
7
2
1700
1600
1500
1400
1300
wavenumber [cm-1
]
0.0
1700
1600
1500
1400
1300
wavenumber [cm-1]
FIGURE 11 Calculated VCD spectra of
6
(R)‐butylone
hydrochloride conformers (top), their average spectrum (middle),
and the experimental spectrum of (R)‐butylone hydrochloride and
VCD noise spectrum (bottom; offset for clarity)
FIGURE 10 Calculated IR absorption spectra of 6 (R)‐butylone
hydrochloride conformers (top), their average spectrum (middle),
and the experimental spectrum of (R)‐butylone hydrochloride
(bottom)
TABLE 3 Experimental and calculated (B3LYP/6‐311++G(d,p))
band frequencies and signs in the VCD spectra of (R)‐butylone
hydrochloride
TABLE 2 Individual IR vibrational modes in the experimental
and calculated (B3LYP/6‐311++G(d,p)) spectra of (R)‐butylone
hydrochloride
Band
Experimental
Calculated
Spectrum, cm−1
Band
Sign
Number Spectrum, cm−1
Experimental Theoretical
1
2
3
4
1630
1604
1494
1458
1625
1600
1490
−
+
+
Band
Spectrum,
Number cm−1
Spectrum,
cm−1
Vibrational Mode
1
2
3
4
1673
1618
1606
1506
1670
1630
1609
1517
ν (C═O)
1467
1454
−
−
ν (C═C), Phe
δ (N―H)
5
6
7
8
9
1443
1409
1356
1300
1264
1438
1396
1365
1337
1252
+
+
+
+
−
δ (C―H),
CH2―CH3,
O―CH2―O
5
6
1494
1452
1499
1452
δ (C―H)
δ (C―H), δ (C═C),
Phe
7
8
1396
1356
1398
1366
δ (C―H), CH2―CH3
configuration of butylone 1 and butylone 2 as (S)‐ and (R)‐
enantiomers, respectively. While ECD reflects electronic
transitions within chromophores (the aromatic ring of
the butylone molecule), VCD is sensitive to local
δ (C―H), δ (C═C),
Phe
9
1265
1262
δ (C═C), Phe

10
SPÁLOVSKÁ ET AL.
8x109
6x109
4x109
2x109
(R)-I
(R)-II
(R)-III
(R)-IV
(R)-V
(R)-VI
0
B3LYP/6-311++G(d,p)
5x109
14
4x109
3x109
2x109
15
12
11
9
2
13
1x109
0
7
10
8
4
1
3
6
5
experimental
14
5x108
15
12
4x108
3x108
2x108
11
9
7
10
4
8
6
13
3
2
5
FIGURE 12 Calculated Raman spectra
of 6 (R)‐butylone hydrochloride
conformers (top), their average spectrum
(middle), and the experimental spectrum
of (R)‐butylone hydrochloride (bottom)
108
0
1
600
800
1000
wavenumber [cm-1
1200
1400
1600
]
vibrational transitions (the carbonyl and amine groups,
aliphatic moieties).40-42 Therefore, the combination of
these two chiroptical methods provided much more
detailed structural information about the studied mole-
cule and, thus, enabled us to reliably assign the absolute
configuration.
determine the 3D structure of the 6 stable lowest energy
conformers with relative abundances of 37%, 24%, 21%,
10%, 7%, and 1% (based on the Boltzmann distribution
at ambient temperature). All used spectroscopic methods
provided
a
very good agreement between the
experimental and the corresponding simulated spectra.
Thus, we believe that this approach can be helpful for
future pharmaceutical, forensic, and molecular‐biology
studies of NPS.
4 | CONCLUSIONS
In this work, we optimized a chiral chromatographic sep-
aration applicable for the enantiomer purity analysis of
butylone, one of the cathinone‐based psychoactive sub-
stances. The separation process may be scaled up for the
chiral separation of several milligrams of a racemic com-
pound in one run. Subsequently, spectroscopic methods
were used for a detailed structural analysis of both
butylone enantiomers. As far as we are concerned, our
study is the first to use chiroptical methods, specifically
ECD and VCD, supported by conventional IR, UV, and
Raman spectroscopies in combination with ab initio cal-
culations at the B3LYP/6‐311++G(d,p) level for the sys-
tematic structural analysis of NPS. Using such powerful
tools, we were able to successfully elucidate the absolute
configuration of both butylone enantiomers and to
ACKNOWLEDGMENTS
The work was supported by the Ministry of the Interior of
the Czech Republic (MV0/VI20172020056). Partial sup-
port 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 Specific
University Research (MSMT No. 20‐SVV/2017 and 20‐
SVV/2018).
ORCID
Dita Spálovská http://orcid.org/0000-0001-9503-6329
František Králík http://orcid.org/0000-0002-5214-3367

SPÁLOVSKÁ ET AL.
11
14. Simmler L, Buser T, Donzelli M, et al. Pharmacological charac-
terization of designer cathinones in vitro. Br J Pharmacol.
2013;168:458‐470.
Michal Kohout http://orcid.org/0000-0003-1447-4453
Bronislav Jurásek http://orcid.org/0000-0002-0262-0065
Lucie Habartová http://orcid.org/0000-0003-0973-0635
Martin Kuchař http://orcid.org/0000-0002-7616-6352
Vladimír Setnička http://orcid.org/0000-0002-9615-7955
15. Berova N, Polavarapu PL, Nakanishi K, Woody RW (Eds).
Comprehensive Chiroptical Spectroscopy: Applications in Stereo-
chemical Analysis of Synthetic Compounds, Natural Products,
and Biomolecules. New Jersey: John Wiley & Sons, Inc.;
2012:872.
REFERENCES
16. Bouř P, Navrátilová H, Setnička V, Urbanová M, Volka K. (3R,4S)‐
4‐(4‐fluorophenyl)‐3‐hydroxymethyl‐1‐methylpiperidine: confor-
mation and structure monitoring by vibrational circular
dichroism. J Org Chem. 2002;67:161‐168.
1. European Monitoring Centre for Drugs and Drug Addiction.
European Drug Report 2016: Trends and Developments.
Luxembourg: Publications Office of the European Union:2016, 84.
2. Fojtíková L, Holubová B, Kuchař M. New psychoactive sub-
stances. Chem listy. 2017;111:234‐238.
17. Julínek O, Setnička V, Řezáčová A, Dohnal J, Vosátka V,
Urbanová M. Product of alaptide synthesis: determination of the
absolute configuration. J Pharm Biomed Anal. 2010;53:958‐961.
3. Jurásek B, Kuchař M. Methoxfenidin. Drugs and Forensics
Bulletin. 2017;23:3‐7.
18. Tománková Z, Setnička V, Urbanová M, et al. Conformational
4. Páleníček T, Lhotková E, Žídková M, et al. Emerging toxicity of
5,6‐methylenedioxy‐2‐aminoindane (MDAI): pharmacokinetics,
behaviour, thermoregulation and LD50 in rats. Prog
Neuropsychopharmacol Biol Psychiatry. 2016;69:49‐59.
flexibility of Corey lactone derivatives indicated by absorption
and vibrational circular dichroism spectra.
J Org Chem.
2004;69:26‐32.
19. Covington CL, Nicu VP, Polavarapu PL. Determination of the
absolute configurations using exciton chirality method for vibra-
tional circular dichroism: right answers for the wrong reasons? J
Phys Chem A. 2015;119:10589‐10601.
5. Hájková K, Jurásek B, Sýkora D, Páleníček T, Mikšátková P,
Kuchař M. Salting‐out assisted liquid‐liquid extraction as a suit-
able approach for determination of methoxetamin in large sets of
tissue samples. Anal Bioanal Chem. 2016;408:1171‐1181.
20. Covington CL, Raghavan V, Smuts JP, Armstrong DW,
Polavarapu PL. Absolute configuration of an axially chiral sulfo-
nate determined from its optical rotatory dispersion, electronic
circular dichroism, and vibrational circular dichroism spectra.
Chirality. 2017;29:670‐676.
6. Shevyrin V, Kupriyanova O, Lebedev AT, et al. Mass
spectrometric properties of N‐(2‐methoxybenzyl)‐2‐(2,4,6‐
trimethoxyphenyl)ethanamine (2,4,6‐TMPEA‐NBOMe), a new
representative of designer drugs of NBOMe series and deriva-
tives thereof. J Mass Spectrom. 2016;51:969‐979.
21. Stephens PJ, Devlin FJ. Determination of the structure of chiral
molecules using ab initio vibrational circular dichroism spectros-
copy. Chirality. 2000;12:172‐179.
7. Concheiro M, Anizan S, Ellefsen K, Huestis MA. Simultaneous
quantification of 28 synthetic cathinones and metabolites in
urine by liquid chromatography‐high resolution mass spectrom-
etry. Anal Bioanal Chem. 2013;405:9437‐9448.
22. Junior FMS, Covington CL, de Albuquerque ACF, et al. Absolute
configuration of (−)‐centratherin, a sesquiterpenoid lactone,
defined by means of chiroptical spectroscopy. J Nat Prod.
2015;78:2617‐2623.
8. Kelly JP. Cathinone derivatives: a review of their chemistry,
pharmacology and toxicology. Drug Test Anal. 2011;3:439‐453.
9. López‐Arnau R, Martínez‐Clemente J, Pubill D, Escubedo E,
Camarasa J. Comparative neuropharmacology of three
psychostimulant cathinone derivatives: butylone, mephedrone
and methylone. Br J Pharmacol. 2012;167:407‐420.
23. Fagan P, Kocourková L, Tatarkovič M, et al. Cocaine hydrochlo-
ride structure in solution revealed by three chiroptical methods.
ChemPhysChem. 2017;18:2258‐2265.
24. Freedman TB, Dukor RK, van Hoof PJCM, Kellenbach ER,
Nafie LA. Determination of the absolute configuration of (−)‐
mirtazapine by vibrational circular dichroism. Helv Chim Acta.
2002;85:1160‐1165.
10. Eshleman AJ, Wolfrum KM, Hatfield MG, Johnson RA,
Murphy KV, Janowsky A. Substituted methcathinones differ in
transporter and receptor interactions. Biochem Pharmacol.
2013;85:1803‐1815.
25. Polavarapu PL, Donahue EA, Shanmugam G, et al. A single chi-
roptical spectroscopic method may not be able to establish the
absolute configurations of diastereomers: dimethylesters of
hibiscus and garcinia acids. J Phys Chem A. 2011;115:5665‐5673.
11. Pauk V, Žihlová V, Borovcová L, Havlíček V, Schug K, Lemr K.
Fast separation of selected cathinones and phenethylamines by
supercritical fluid chromatography.
J
Chromatogr A.
2015;1423:169‐176.
26. Urbanová M, Setnička V, Volka K. Measurements of concentra-
tion dependence and enantiomeric purity of terpene solutions as
a test of a new commercial VCD spectrometer. Chirality.
2000;12:199‐203.
12. Ondra P, Válka I, Knob R, Ginterová P, Maier V. Analysis of
amphetamine‐derived designer drugs by gas chromatography
with mass spectrometry. J Anal Toxicol. 2016;40:78‐85.
13. Power JD, McGlynn P, Clarke K, McDermott SD, Kavanagh P,
O'Brien J. The analysis of substituted cathinones. Part 1: chemi-
cal analysis of 2‐, 3‐ and 4‐methylmethcathinone. Forensic Sci
Int. 2011;212:6‐12.
27. Tatarkovič M, Synytsya A, Šťovíčková L, et al. The minimizing of
fluorescence background in Raman optical activity and Raman
spectra of human blood plasma. Anal Bioanal Chem.
2015;407:1335‐1342.

12
SPÁLOVSKÁ ET AL.
28. Člupek M, Matějka P, Volka K. Noise reduction in Raman spec-
tra: finite impulse response filtration versus Savitzky–Golay
smoothing. J Raman Spectrosc. 2007;38:1174‐1179.
37. Bouř P, Maloň P. The MCM Program. Academy of Sciences:
Prague, Czech Republic; 1995−2009.
38. Polavarapu PL, Covington CL. Comparison of experimental and
calculated chiroptical spectra for chiral molecular structure
determination. Chirality. 2014;26:539‐552.
29. Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian 09, D.01;
Wallingford CT, 2009.
30. Covington CL, Polavarapu PL. CDSpecTech: a single software
suite for multiple chiroptical spectroscopic analyses. Chirality.
2017;29:178‐192.
39. Debie E, De Gussem E, Dukor RK, Herrebout W, Nafie LA,
Bultinck P. A confidence level algorithm for the determination
of absolute configuration using vibrational circular dichroism
or Raman optical activity. ChemPhysChem. 2011;12:1542‐1549.
31. Wolrab D, Frühauf P, Moulisová A, et al. Chiral separation of
new designer drugs (cathinones) on chiral ion‐exchange type sta-
tionary phases. J Anal Bioanal Chem. 2016;120:306‐315.
40. Gorecki M. A configurational and conformational study of (−)‐
oseltamivir using a multi‐chiroptical approach. Org Biomol
Chem. 2015;13:2999‐3010.
32. Weiß JA, Mohr S, Schmid MG. Indirect chiral separation of new
recreational drugs by gas chromatography‐mass spectrometry
using trifluoroacetyl‐l‐prolyl chloride as chiral derivatization
reagent. Chirality. 2015;27:211‐215.
41. Polavarapu PL. Why is it important to simultaneously use more
than one chiroptical spectroscopic method for determining the
structures of chiral molecules? Chirality. 2008;20:664‐672.
33. Merola G, Fu H, Tagliaro F, Macchia T, McCord BR. Chiral sep-
aration of 12 cathinone analogs by cyclodextrin‐assisted capillary
electrophoresis with UV and mass spectrometry detection. Elec-
trophoresis. 2014;35:3231‐3241.
42. Nicu VP, Mándi A, Kurtán T, Polavarapu PL. On the complemen-
tarity of ECD and VCD techniques. Chirality. 2014;26:525‐531.
SUPPORTING INFORMATION
34. Schwaninger AE, Meyer MR, Maurer HH. Chiral drug analysis
using mass spectrometric detection relevant to research and
practice in clinical and forensic toxicology. J Chromatogr A.
2012;1269:122‐135.
Additional Supporting Information may be found online
in the supporting information tab for this article.
35. Taschwer M, Grascher J, Schmid MG. Development of an
enantioseparation method for novel psychoactive drugs by
HPLC using a Lux Cellulose‐2 column in polar organic phase
mode. Forensic Sci Int. 2017;270:232‐240.
How to cite this article: Spálovská D, Králík F,
Kohout M, et al. Structure determination of
butylone as a new psychoactive substance using
chiroptical and vibrational spectroscopies. Chirality.
2018;1–12. https://doi.org/10.1002/chir.22825
36. Berrang BD, Lewin AH, Carroll FI. Enantiomeric α‐
aminopropiophenones (cathinone): preparation and investiga-
tion. J Org Chem. 1982;47:2643‐2647.