Comprehensive analytical characteristics of N-(adamantan-1-yl)-1- (cyclohexylmethyl)-1H-indazole-3-carboxamide (ACHMINACA)

The aim of this study was to clarify the most essential analytical features of N-(adamantan-1-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (one of the newest cannabimimetics called ACHMINACA), to make them useful for analytical identification of this compound. The compound was analyzed by gas chromatography–mass spectrometry, high-resolution liquid chromatography–mass spectrometry, crystal X-ray diffraction and spectroscopic methods such as nuclear magnetic resonance, Raman, and infrared spectroscopies. Detailed and comprehensive analytical data have been acquired for ACHMINACA. Although brief descriptions of the partial data of ACHMINACA have appeared recently, this article provides the most detailed and comprehensive analytical data of ACHMINACA to our knowledge. Our data will significantly broaden the knowledge about the compound structure extending the possibility of its orthogonal analysis. The gathered data are useful for forensic, toxicological, and clinical purposes.


Introduction
A characteristic feature of the contemporary market of illegal substances is the huge supply of novel psychoactive compounds. Hundreds of them have been registered over the last 10 years by specialized European and United Nations offices, such as the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime [1,2]. Among them, synthetic cannabinoids constitute one of the most numerous and widespread groups. Their structural evolvement started with the modification of compounds containing the cyclohexylphenol or arylindole ketone core [3,4]. Over the course of 10 years, many derivatives of these compounds have appeared on the market, e.g., indole-and indazole carboesters, indole-and indazole carboxamides, indole-2,2,3,3-tetramethylcyclopropyl ketones, pyrazole carboxamides and other groups [5][6][7][8][9]. A multitude of substituents meeting the requirements for the cannabimimetic activity that can be attached to the enlisted core scaffolds and possible regioisomerism [4,[10][11][12] further expand the structural variety of synthetic cannabinoids. Until recent years, synthetic cannabinoids with adamantyl (tricyclo [3.3.1.13,7]dec-1-yl) moiety (e.g. AB-001, APICA, APINACA, APINAC, and AMPPPCA [8,[12][13][14][15][16][17]) have been more and more frequently available on the black market of illegal substances which has increased a need for new reliable methods of their detection.
This article presents comprehensive analytical characteristics of a novel synthetic cannabinoid seized by Polish law enforcement authorities-ACHMINACA, N-(adamantan-1-yl)-1-(cyclohexylmethyl)-1H-indazole-3-carboxamide (see Fig. 1). This analog of APINACA, with the cyclohexylmethyl group in place of the pentyl chain, was examined using the following methods: gas chromatography coupled with mass spectrometry (GC-MS); attenuated total reflection (ATR) Fourier-transform infrared (FTIR) spectroscopy; nuclear magnetic resonance (NMR) spectroscopy; liquid chromatography with ultraviolet-visible (UV-VIS) photodiode array (PDA) and with mass spectrometry detection (LC-PDA-MS); and powder and single crystal X-ray diffraction. To the best of our knowledge, complete structural characterization of ACHMINACA has not been reported yet in scientific literature; only a few spectroscopic data are available from SWGDRUG and Slovenian Police reports [18,19].

Materials
Acetonitrile and methanol (all LC-MS grade) were purchased from the Polish Chemical Plant POCh (Gliwice, Poland); formic acid from Sigma-Aldrich (Seelze, Germany); deuterated chloroform from Armar AG (Döttingen, Switzerland); the Milli-Q system from Millipore (Millipore, Bedford, MA, USA) applied for water purification. ACHMI-NACA sample was donated by the forensic laboratory of the

GC-MS analysis
Qualification of the compound in the prepared sample was performed using GC/MS QP2010 (Shimadzu, Kyoto, Japan). A ZB5-MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness; Phenomenex, Torrance, CA, USA) was used. Helium (grade 5.0) was used as a carrier gas. One microliter of the sample was injected by an AOC-20i type autosampler (Shimadzu). The injector's temperature was 310 °C. The following temperature program was applied: start at 50 °C, a linear temperature increase up to 315 °C at the rate of 7 °C × min −1 and hold for 20 min. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV; the ion source temperature was 220 °C. The mass spectra were measured in the range 35-400 amu. Qualitative analysis was carried out comparing the retention indices and MS spectra for the obtained peaks with the analogous data from the NIST14 database. Additionally, the retention index of the component was calculated according to [20].

LC-MS analysis
The chromatographic measurements were performed on a high-resolution (HR) LC-MS system consisting of a ultrahigh-performance liquid chromatography (UHPLC) instrument (UltiMate 3000; Dionex, Sunnyvale, CA, USA), a linear trap quadrupole-Orbitrap mass spectrometer (LTQ-Orbitrap Velos from Thermo Fisher Scientific, San Jose, CA, USA) and an electrospray ionization (ESI) source. A Gemini C18 column (100 × 4.6 mm i.d., 3 μm particle size; Phenomenex) was employed for chromatographic separation performed using isocratic elution (Phenomenex pre-column, 3 × 4 mm i.d, 3 μm particle size, was used for column protection). The mobile phase was acetonitrile/water (75:25, v/v). The total run time was 45 min at the mobile phase flow rate of 0.5 mL/min. In the course of each run, PDA spectra in the range of 190-600 nm and MS spectra in the range of m/z 100-1000 were collected continuously. The ESI source was operated in the positive mode under the following specific conditions: spray voltage, 3.5 kV; sheath gas, 40 arbitrary units; auxiliary gas, 10 arbitrary units; sweep gas, 10 arbitrary units; and capillary temperature, 320 °C. Nitrogen (99.98%) was employed as the sheath, auxiliary and sweep gases. The scan cycle was used for a full-scan event at the resolution of 60,000.

MS analysis with direct flow injection
The solution of the examined compound in methanol (0.1 mg/mL) was directly introduced at the rate of 100 µL/ min to the ion source of the ion-trap mass spectrometric system (LCQ Advantage Max; Thermo Finnigan, San Jose, CA, USA). The ESI needle potential was 4.5 kV in the positive ionization mode. The function of secondary ion fragmentation (MS 2 ) was applied for the value at m/z 392. The collision energy was 25%.

ATR-FTIR
The infrared spectrum of the examined compound was registered using a Nicolet™iS50 FTIR spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with ATR crystal, employing the spectral range from 4000 to 400 cm −1 . The spectrum of the obtained solid sample was acquired without any sample pretreatment.

Raman spectroscopy
Acquisition of Raman spectrum for the ACHMINACA powder sample was performed using inVia Reflex Raman microscope (Renishaw, Wotton-Under-Edge, Gloucestershire, UK) equipped with a 785 nm semiconductor laser. The data were collected in the wavenumber range from 500 to 3500 cm −1 .

NMR spectroscopy
For the NMR analysis, 10 mg of the seized material was dissolved in 0.5 mL of CDCl 3 , centrifuged and transferred to a 5 mm NMR tube. The acquisition of NMR spectra was performed using an Ascend 600 spectrometer (Bruker, Bremen, Germany). The following NMR techniques were employed to elucidate the structure of the examined compound and to assign the NMR signals: -1D: 1 H, 13  The chemical shifts of 1 H and 13 C nuclei were calibrated using the internal reference signals (tetramethylsilane: TMS).

Powder X-ray diffraction
The powder diffraction measurements of the seized material were performed using an Empyrean diffractometer (PANalytical, Malvern, UK) employing Cu anode (voltage: − 40 kV; current: 25 mA) and 2θ range from 4.96° to 90.03° with the step size of 0.026°.

Single crystal diffraction
The single-crystal data were collected at 20 °C using a Rigaku XtaLAB diffractometer with a MicroMax-007 HF rotated anode X-ray source and Pilatus 200 K surface detector (Rigaku Corporation, Tokyo, Japan). The CrysA-lisPro software (Rigaku Corporation) was used for the collection, integration, and analysis of the obtained data. The crystal structure was solved by direct methods using the SHELXS-97 program and refined using the SHELXL-97 program [21].  Fig. 3a was similar to that for ACHMINACA described in the SWGDRUD database [18]. The estimated retention index equaled 3081.

LC-MS and direct flow injection ESI-MS 2
The exemplary LC-MS chromatogram of ACHMINACA methanolic solution is shown in Fig. 2b, whereas HR-MS 1 3 data corresponding to this compound is gathered in Table 1. The obtained data indicated the following molecular weight of ACHMINACA [M + H] + ion and its elemental composition: 392.27011 Da and C 25 H 33 N 3 O, respectively. The very low difference between the theoretical and the experimental mass (Δppm) for ACHMINACA ions proved the correctness of the elemental analysis.
The m/z values and intensities of individual product ions obtained by direct injection of ACHMINACA solution into the ion source of the ion-trap mass spectrometer are also collected in Table 1. The three most intensive fragment ions at m/z 134.8, 257.5, and 258.7, indicate the similarity between ESI and EI fragmentation patterns (see Fig. 4).

IR, Raman, and UV-VIS spectroscopies
The IR spectrum of the examined compound is shown in Fig. S1 (see Supplementary material). The following characteristic bands could be distinguished for ACHMINACA: -two bands of secondary amide group representing carbonyl stretching and N-H bending modes at 1655 cm −1 and 1524 cm −1 , respectively; -weak bands of N-H stretching around 3400 cm −1 ; -asymmetric aromatic ring stretching vibrations appearing at 1490 cm −1 along with out-of-plane bending of aromatic ring and C-H bonds modes giving rise to the signals in the region of 800 to 700 cm −1 . According to the signal of the PDA detector, two maxima (at 210 nm and at 302 nm) were observed on the UV-Vis spectrum of ACHMINACA (see Fig. S3).

NMR spectroscopy
The 1 H and 13 C spectra of ACHMINACA in CDCl 3 are shown in Figs. S4 and S5, respectively. The structural assignment of 1 H and 13 C signals was conducted on the basis of 2D correlation spectra and 1D s-TOCSY technique used for the overlapping high field signals (see Figs. S6-S12). For the TOCSY, selective excitations of 4.17, and 2.23 ppm 1 H nuclei were employed, generating the 1D spectra showing only signals from cyclohexylmethyl and adamantyl proton spin systems, respectively (see Fig. S10 and S11). The structural assignment for nuclei of the cyclohexyl group was additionally facilitated using the refocused 1,1-ADEQUATE technique optimized for CH n groups, with n greater than 1. Its signals gave direct information about two bond 1 H-13 C connectivities and indirect information about one bond 13 C-13 C connectivities (Fig. S12). Table 2 presents the description of 1 H and 13 C signals and the most important correlations from 2D spectra.

X-ray diffraction
The seized material was analyzed with powder X-ray diffraction (see Fig. S13). Following parameters were determined on the basis of powder data: the crystal system -monoclinic; space group -P2 1   For the crystal structure solution, a single crystal measurement was conducted. Two independent ACHMINACA conformers (see Fig. 5) were observed in the crystal net (see Fig. S14). The single-crystal data and parameters from structure refinement are presented in Table 3.

Discussion
Spectroscopic techniques are fundamental for the identification of new psychoactive substances. In the case of ACHMINACA, only the most basic spectra like FTIR, EI-MS, and 1 H NMR were reported by a few institutions [18,19]. Analogous data were collected in our laboratory and stay in good agreement with the reports cited above. Additional techniques of ESI-MS (Fig. 4), Raman (Fig.  S2) and UV-VIS (Fig. S3) spectroscopies were employed in the present study for further structural characterization of ACHMINACA allowing to extend the identification possibility of the most important structural ACHMINACA features, e.g., the presence of the heteroaromatic core and of the secondary amide group.
Although the discrimination of 1-adamantyl and 2-adamantyl isomers of other synthetic cannabinoids using GC-MS was reported [22], no similar data are available for ACHMINACA. For this reason, 2D NMR techniques ( Table 2, Figs. S6-S12) were applied for the precise determination of ACHMINACA regioisomerism. 1 H and 13 C signals assigned for aromatic nuclei and their correlations resembled the resonance patterns of typical 1H-indazole carboxamides. Moreover, NOESY correlation between H7 and H1a clearly implied that the methyl cyclohexyl substituent is attached at position 1 of the aromatic moiety. The NOESY and COSY correlations of amide and adamantyl protons indicated the quaternary character of the adamantyl carbon atom connected to the amide nitrogen.
Although the techniques of molecular spectroscopy are the first choice for the structural characterization of novel designer drugs, X-ray diffraction methods may also be employed because most of the materials containing  new illegal substances have a solid form with a developed crystalline structure. The seized sample of ACHMI-NACA was analyzed by powder and single-crystal X-ray diffractometers without any sample pretreatment. The former technique allowed to determine the space group and lattice parameters of the examined material, but the crystal structure was not possible. A single crystal was successfully separated from the crude sample and examined by single-crystal diffraction giving the values of unit cell parameters similar to those in powder diffraction ( Table 3). The obtained structure consisted of two ACHMINACA conformers differing only in the orientation of cyclohexyl moiety (Fig. 5). Both X-ray diffraction methods can be successfully utilized for the noninvasive characterization of materials containing synthetic cannabinoids. The single crystal experiment may even be used as a stand-alone technique for the purpose of complete identification replacing the long analytical process employing a wide array of molecular spectroscopy techniques.

Conclusions
A C H M I N A C A , t h e N -( a d a m a n t a n -1 -y l ) -1-(cyclohexylmethyl)-1H-indazole-3-carboxamide, is one of the novel synthetic cannabinoids, an analog of API-NACA with the cyclohexylmethyl group in place of the pentyl chain. This study presented the complete structural characterization of this psychoactive compound obtained by GC-MS, ATR-FTIR spectroscopy, Raman spectroscopy, 1D and 2D NMR, LC--PDA-MS, and powder and single-crystal X-ray diffraction. The results not only confirm the scantily provided data concerning ACHMINACA, but also significantly broaden the knowledge about the compound structure and extending the possibility of its orthogonal analysis.