Spectroscopic and crystallographic characterization of a new cathinone derivative: 1-phenyl-2-(butylamino)hexan-1-one hydrochloride (N-butylhexedrone)

In this study, a new cathinone derivative, N-butylhexedrone, emerged on new psychoactive substances (NPS) market in Poland was described and characterized. The compound was analyzed by gas chromatography—mass spectrometry, X-ray crystallography and infrared, Raman, ultraviolet-visible and nuclear magnetic resonance spectroscopic approaches. We confirmed the presence of the compound in the seized material and obtained detailed and comprehensive physicochemical characterization of N-butylhexedrone—new cathinone derivative available on the NPS market. In this study, we presented chromatographic, spectroscopic and crystallographic characterization of a new cathinone derivative that emerged on the NPS market in 2019. The obtained analytical data should be useful for forensic and toxicological purposes in quick and reliable compound identification.


Introduction
During the last decade, new psychoactive substances (NPS) have become a serious social and legal issue; their widespread availability translates into a serious threat to health and life of humans abusing them. Among compounds that dominate on the present NPS market are synthetic cathinones; their mode of action involves stimulation similar to that exerted by amphetamine or cocaine. There are numerous reports of cathinone derivatives' effects and overdosing case reports [1][2][3][4][5][6].
Synthetic cathinones are a widespread class of compounds present for years on the NPS market, but successive chemical modifications make them an analytical challenge for toxicologists, medics or law enforcement officers. In this report, we present physicochemical characteristics of N-butylhexedrone hydrochloride (Fig. 1)-a novel derivative identified in the evidence material seized by police officers at the beginning of 2019 in Poland. The study results presented herein are a continuation of our previous reports concerning NPS and could be used for rapid and unequivocal identification of a given compound in crime investigation and toxicology laboratories [7,8]. Of particular relevance are crystallographic studies, because knowledge of cell parameters for a compound allows for its fast noninvasive identification without the need for preliminary sample preparation. Because the material that has emerged on the NPS market and was seized contains very often crystal particles, generated crystallographic data may be further used for identification [9][10][11].
The data for characterizing the substances were obtained by gas chromatography-mass spectrometry (GC-MS), direct infusion electrospray ionization mass spectrometry (ESI-MS), infrared (IR), Raman and ultraviolet-visible (UV-VIS) spectroscopies, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. To our knowledge, this is the first report that identifies in the seized material and characterizes N-butylhexedrone in detail circulated on the NPS market. Some data for this compound are available in the report from Slovenian National Forensic Laboratory [12].

Chemicals
In our study, all reagents used were of the HPLC or MS purity grade. Water (Chromasolv), methanol, isopropanol and deuterated chloroform (CDCl 3 for NMR analysis) were purchased from Sigma-Aldrich (Poznań, Poland).

Sample preparation
The sample was provided by drug enforcement agencies as material seized on the illicit drug market and was in pure powdered form. For the purpose of GC and ESI-MS, the 10-mg sample was dissolved in 1-mL methanol without the need for ultrasonication. An aliquot of 10 µL was collected from the solution and diluted 100-fold with methanol and analyzed by GC-MS and ESI-MS. For NMR spectroscopic analysis, 10 mg of the powdered sample was dissolved in 0.6 mL CDCl 3 . For the IR, Raman and UV-VIS purpose, the 5-mg aliquot sample was taken for analysis. The IR and Raman analyses were performed without any further sample treatment, and for the UV-VIS analysis, the samples were dissolved and diluted in methanol.

GC-MS analysis
For GC-MS analysis, the Thermo Trace Ultra chromatograph was used, coupled with the Thermo DSQ mass spectrometer (Thermo Scientific, Waltham, MA, USA). The analyses were carried out with use of the Rxi ® -5Sil MS column (Restek, Bellefonte, PA, USA). The following working parameters were employed: injector temperature, 260 °C; oven temperatures, 100 °C for 2 min, ramp at 20 °C/min to 260 °C; the carrier gas (helium) flow rate, 1.2 mL min −1 ; MS transfer line temperature, 250 °C; MS source temperature, 250 °C; the injection volume, 1 μL, the splitless mode.

Direct infusion electrospray ionization mass spectrometry
Thermo TSQ Vantage mass spectrometer with electrospray ionization source (Thermo Scientific) was used. The following working parameters for the direct infusion ESI-MS experiment were employed: sheath gas pressure, 5 psi; heated (H)-ESI vaporizer temperature, 50 °C; spray voltage, 3500 V; ion transfer tube temperature, 50 °C; direct infusion syringe flow rate, 5 μL/min. The obtained data were processed using Xcalibur and TSQTune software (Thermo Scientific). The analytes were electrosprayed in the positive mode (ESI(+)-MS). Fragmentation in the ESI-MS 2 mode was carried out in the scanning range of m/z 50-255. The ESI-carrier and collision gases were nitrogen and argon, respectively.

NMR spectroscopy
The NMR spectra of the samples were recorded with use of the UltraShield 400 MHz apparatus (Bruker, Bremen, Germany), and CDCl 3 was used as a solvent. The data were collected with the chemical shift referenced to a residual solvent signal.

Fourier transform infrared (FT-IR), Raman and UV-VIS spectroscopies
The IR spectrum of the powder evidence material was acquired with use of the Nicolet iS50 FT-IR spectrometer (Thermo Scientific), using the attenuated total reflection technique, and the spectrum was collected in the wave number range 3500-400 cm −1 . Raman measurements were taken using a Thermo Scientific™ DXR™2xi Raman imaging microscope. The data were collected using a 780 nm laser. The UV-VIS absorption spectrum was recorded in the methanol solution using the Thermo Scientific Evolution 160 UV-VIS spectrophotometer, and the spectrum was collected in range of wavelength 190-400 nm. The UV-VIS absorption spectrum was recorded in quartz cuvette with 1 cm light path length.

X-ray spectroscopy
The single-crystal X-ray experiments were performed at 100 K. The data were collected using a SuperNova kappa diffractometer with Atlas charge coupled device detector (Agilent Technologies, Santa Clara, CA, USA). For the integration of the collected data, the CrysAlis Pro software (version 1.171.38.41q, 2015; Rigaku Oxford Diffraction, Rigaku, Tokyo, Japan) was used. The structures were solved using direct methods with the SHELXS-2013 software, and the solutions were refined using SHELXL-2018/3 program [13]. CCDC 1963389 and CCDC 1963390 contain supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via: www.ccdc.cam.ac.uk/data_reque st/cif.

GC-MS and ESI-MS
The sample was analyzed by GC-MS, and the resulting mass spectrum of the compound is shown in Fig. 2.
In the mass spectrum obtained in the EI-MS mode, one main fragment ion was detected at m/z 142. Other less intense fragments present in the spectrum were m/z 77, 86 and 105. Possible structures of the fragmentation products derived from the parent structure of the investigated compound are presented in Fig. 3, which is in accordance with fragmentation pathways proposed in the literature [14]. The obtained spectrum is also identical with that originating from the report of Slovenian National Forensic Laboratory [12]. When compared with the mass spectrum of NEH (N-ethylhexedrone) [8], it can be noticed that there are the same peaks present at m/z 77 and 105, and the spectrum differs only in the base peak found at m/z 114 for NEH. The difference of 28 Da between m/z 114 (NEH) and m/z 142 (N-butylhexedrone) corresponds well with the presence of two additional methylene groups in the investigated compound. This suggest the bond cleavage between carbon 1 and 2 (carbon numbering shown in Fig. 1) while the fragmentation. Also, it can be noticed that the difference of 28 Da can be observed in peaks found at m/z 58 for NEH and at m/z 86 for N-butylhexedrone.
In the ESI-MS spectrum, the protonated molecule [M + H + ] was seen at m/z 248 (Fig. S1). The sample was directly infused to the ion source. The sample was also analyzed in the MS/MS mode (Fig. S2). In the MS/ MS mode, elimination of water molecule was observed [M + H + -H 2 O], which is characteristic for certain cathinone derivatives [15,16]. Compound optimization for selected reaction monitoring experiments was also performed. Three most abundant peaks were found at m/z 91, 118 and 174. The probable fragmentation pathways are presented in Fig. 4.

H and 13 C NMR spectroscopy
The NMR spectroscopy was employed to confirm the structure of the investigated compound (Figs. S3 and S4). The data for N-butylhexedrone are presented in Table 1 (atom numbering according to Fig. 1).

IR, Raman, UV-VIS spectra and melting point
IR and Raman techniques can be useful for characterizing NPS, especially during quick identification and profiling (fingerprinting). In the IR spectrum (Fig. S5), a strong carbonyl stretch at 1687 cm − 1 was observed. Aliphatic and aromatic C-H stretching at 2700-3000 cm − 1 can be observed as well. In the Raman spectroscopy (Fig. S6), strong C = O band was seen at 1691 cm − 1 . The C-C aromatic bands lay actually at the same wave number values as in the IR spectrum. The UV spectra recorded by UV-VIS spectrometry showed absorption maxima at 202 and 251 nm for the investigated compound (Fig. S7). Melting point of the N-butylhexedrone hydrochloride measured in the classical way was 138-140 °C.

X-ray studies
Compound 1 formed orthorhombic crystals in the P2 1 2 1 2 1 space group (form A) or monoclinic crystals in the P2 1 /c space group (form B) when crystallized from dimethyl sulfoxide or ethyl alcohol, respectively. In the orthorhombic crystals, all molecules occur as the R-isomers. In the monoclinic crystals, both enantiomers occur together with additional ethyl alcohol and water molecules in the unit cell. Crystal data and structure refinement for both crystals are summarized in  : 150.50-169.55 o ), respectively. These values are very similar to the data reported for other cathinone derivatives [8]. Torsion angle C7C12N1C13 was 73.70 o in R-isomer of form A. Torsion angles C7C12N1C13 were identical in both enantiomers (± 71.10 o ) in crystals of form B. In crystals of form A, no weak hydrogen bonds were present between cathinone molecules. In crystals of form B, two molecules: R-and S-isomers, can form pairs in which the π···π interactions between phenyl rings may occur, which is shown in Fig. S8. The phenyl rings are separated from each other by 3.363 Å and centroid distance of 4.076Å (124.23 o angle).
Other short distances occurring in the structure of compound 1: two C-H···Cl and two N-H···Cl short distances, testifying to the presence of weak interactions between molecules in form A are shown in Fig. S9. Calculated C-H12···Cl1 distance (2.572Å; 171.1 o ) and C-H6···Cl1 distance (2.80Ǻ; 169.3 o ) are shorter than sum of van der Waals radii = 2.95Ǻ.

Discussion
Crystallographic methods allow to define precisely the particular components of a sample provided it contains monocrystalline components amenable to mechanical The numbering of carbon atoms is given in Fig. 1 Atom position Carbon chemical shifts (ppm) separation. When the sample presents a mixture and its components have to be separated by chromatographic methods or the sample is an amorphous substance requiring recrystallization in order to obtain monocrystals, there is a concern of incorporating solvent molecules in crystals of a given compound. This situation has occurred in the particular case presented herein. Recrystallization led to the incorporation of solvent molecules (water and ethanol) in the elementary cell during formation of crystals. Our previously performed analyses of directly marketed NPS samples never revealed crystals of cathinone hydrochloride (in one case disulphuric (VI) acid [17]) salts that would also contain solvents in elementary cells. Such cases need to be considered when analyzing unknown samples by crystallographic methods. It should also be mentioned that very recently during preparation of this manuscript a report has appeared [18] dealing with physical and chemical characterization of N-butylhexedrone. These Russian investigators analyzed compound 1 using chromatographic methods coupled with mass spectrometry, and NMR and IR spectroscopies. Their However, in our study we performed ultraviolet-visible spectroscopy and crystallographic analyses which add, in our opinion, some interesting data from the perspective of full characteristics of NPS and which, by far, are underestimated in forensic science. For many unknown compounds which surface on the NPS market, it is feasible to obtain monocrystals from analyzed samples. They would be suitable for crystallographic analyses allowing unequivocal substance identification. In our opinion, co-implementation of crystallographic methods in NPS analyses ought to meet with wider attention from toxicologists and forensic scientists.

Conclusions
In this study, we presented chromatographic, spectroscopic and crystallographic characterization of a new cathinone derivative that emerged on the NPS market in 2019. The obtained analytical data should be useful for forensic and toxicological purposes in quick and reliable compound in the crystal. Ellipsoids correspond to 50% probability levels identification. Our report is linked to CCDC repository entry for this compound, and its characteristics can be found there including elementary cell data, of particular usefulness for quick analysis. Although some data for the investigated compound have been available [12,18], to our knowledge this study provides the first detailed and comprehensive report including X-ray crystallographic data on N-butylhexedrone.