Cumyl‐CBMICA: A new synthetic cannabinoid receptor agonist containing a cyclobutyl methyl side chain

Since the beginning of the phenomenon of new psychoactive substances (NPS), synthetic cannabinoid receptor agonists (SCRAs) have been the largest and most prevalent subclass of these drugs in Europe. Many countries implemented specific legislation scheduling classes of substances defined on the basis of their chemical structure to reduce supply. We describe the identification and analytical characterization within the EU project ADEBAR plus of 1-(cyclobutylmethyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide which resulted in the formal notification through the Early Warning System of the European Monitoring Centre for Drug and Drug Addiction (EMCDDA). This is the first identification of this new SCRA worldwide and the analytical data was distributed (inter-)nationally right after identification in 2019. First, the substance was isolated from the herbal material using preparative high-performance liquid chromatography (HPLC). Structure elucidation and analytical characterization were performed using gas chromatography-mass spectrometry (GC-MS), gas chromatography-solid state infrared spectroscopy (GC-sIR), liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qToF-MS), Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. The new compound contains a cyclobutyl methyl group as a side chain and has not been described in any patent to our knowledge. Based on the semisystematic nomenclature of SCRAs, we propose Cumyl-CBMICA as a short name for the compound.


| INTRODUCTION
Synthetic cannabinoid receptor agonists (SCRAs) were originally synthesized for medical purposes but have become the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) since their first emergence in 2008. 1,2 The structural diversity of SCRAs is huge, although in recent years, the number of new SCRAs hitting the market decreased. Among these new compounds, only a small number gains higher margins. One example is 4F-MDMB-BINACA (Figure 1), which gained international relevance after its first appearance in Sebastian Halter and Benedikt Pulver contributed equally to this study. December 2018. [3][4][5] In Germany, however, this SCRA was already controlled by a specific law on NPS, the NpSG, enacted in November 2016. The NpSG is a generic law that controls groups of substances based on a modular system defined by chemical structures. 6 Concerning SCRAs, the law defines four structural elements: A core structure, which is connected to a linked group by a linker with a defined position, and a side chain attached to a designated nitrogen atom of the core structure. This modular structure was previously proposed by Kikura-Hanajiri et al. 7 A few online shops selling herbal blends containing SCRAs have a particular interest in and emphasize the legality of their products. Only a few weeks after the introduction of the NpSG, these online shops reacted by selling herbal blends with the SCRA Cumyl-PEGACLONE, which carries a γ-carbolinone structure not covered by the law at that time. 8 In addition, substances with core structures like carbazoles and azaindoles appeared on the market. To keep up with these developments, the NpSG was amended in July 2019 by adding several core structures and substitution patterns. Due to this regulation, all SCRAs with considerable prevalence on the drug market at that time were covered by the law. 9 Similar to Cumyl-PEGACLONE (Figure 1), the new synthetic cannabinoid reported in this work is structurally related to Cumyl-PICA ( Figure 1). However, instead of modifying the core structure, this time, the side chain was modified to bypass the NpSG. Although there are no data so far on the pharmacology of such compounds, cannabimimetic activity can be anticipated due to the high variability of the side chain reaching from aliphatic butyl (e.g., JWH-073), pentyl (5F-Cumyl-PINACA 10,11 ), or cyclohexyl methyl (e.g., MDMB-CHMICA 12 ) substituents to unsaturated substituents like fluorobenzyl (e.g., AMB-FUBINACA 13  The generic definitions of the NpSG have been updated, and this newly discovered substance and its analogs are scheduled since the July 9, 2020 (Supporting Information S19).
The structure of Cumyl-CBMICA was elucidated within the EU funded project ADEBAR plus (www.projekt-adebar.eu). The scope of ADEBAR plus is to provide cooperative structural clarification of unknown substances for police and customs forensic laboratories.
Unidentifiable samples are analyzed using a variety of orthogonal analytical methods to acquire a complete set of analytical data for unambiguous structural clarification. All analytical data are swiftly published in print form and electronically importable, universal formats to share with national (police and customs) and international entities (European Drug Monitoring, worldwide Data Hub: www.npsdatahub.com). Each identified substance is subject of an analytical report in which the spectra and results of the characterization are shown as well as any peculiar and notable findings which appeared during the process of acquiring the analytical data. In addition, substances that are identified for the first time in Germany or the EU are immediately reported to the Early Warning System (EWS) of the EMCDDA via the National Focal Point. Upon completion of the analytical process, we reported the comprehensive set of analytical data to the EDND on November 7, 2019. Meanwhile, two seizures of powder containing Cumyl-CBMICA were made in the Netherlands and notified in the EDND on July 2, 2020. 15 2 | METHODS

| Seizure of herbal blends
Several plain, black ziplock bags containing herbal blends were seized by the Bavarian State Police on August 9, 2019. The bags were plain without any logo or artwork only labeled "AK 47 Gold 15 g." Between all bags, a total of around 100 g of plant material was seized.

| Purchasing of herbal blends
Nearly at the same time, in total 14 herbal blends from three different online shops were purchased during online monitoring which has been performed by the Institute for Forensic Medicine in Freiburg continually since 2009. Scientific, Schwerte, Germany), and finally, the supernatant was collected in a separate vial.

| GC-MS method
For gas chromatography-mass spectrometry (GC-MS) analysis of the unknown compound, 10 μl of the extract were transferred into a GCvial and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 100 μl of dried ethyl acetate. One microliter was injected into the GC-MS system. The GC-MS system consisted of a 6890N-series gas chromatograph combined with a 5973-series mass selective detector and a 7683 B series injector (Agilent, Waldbronn, Germany). The software used was Chemstation G1701GA version D.03.00.611 (Agilent, Waldbronn, Germany). The detailed method is described elsewhere. 16 Briefly, carrier gas was helium, injection port temperature was set to 270 C, the flow rate was 1 ml/min, oven temperature was 100 C for 3 min, then ramped to 310 C at 30 K/min, 310 C were kept for 10 min. Electron ionization (EI, 70 eV) was used, and the MS was operated in scan mode (m/z 50 to 550 amu). The obtained mass spectra were compared with commonly used EI-MS spectra libraries (NIST, Wiley, MPW) and to an in-house library of previously identified synthetic cannabinoids. 18% within 0.9 min, and to 50% within another 1.5 min. Flow increased to 0.223 ml/min within this 1.5 min. Solvent B increased to 99% within 11.5 min, whereas the flow increased to 0.400 ml/min. B was held for 2 min, and flow increased to 0.480 ml/min. The initial conditions were then restored within 0.1 min and held for 2.9 min. The flow decreased to 0.20 ml/min within 0.1 min and was held for 0.9 min to re-equilibrate the system. The total runtime was 20 min.

| Preparative HPLC
Two grams of the herbal blend were extracted with 14 ml ACN.

| Gas chromatography solid-state infrared spectroscopy
A GC-solid phase-IR-system consisting of an Agilent GC 7890B (Waldbronn, Germany) with an Agilent G4567A probe sampler and a DiscovIR-GCTM (Spectra Analysis, Marlborough, MA, USA) was used.
Approximately 2 mg of the compound were dissolved in 2 ml CHCl 3 .
The column eluent was cryogenically accumulated on a spirally rotating ZnSe disk cooled by liquid nitrogen. IR spectra were recorded through the IR-transparent ZnSe disk using a nitrogen-cooled mercury-cadmium-telluride (MCT) detector. Data were processed using GRAMS/AI Ver. 9.1 (Grams Spectroscopy Software Suite, Thermo Fischer Scientific, Dreieich, Germany) followed by OMNIC Software, Ver. 7.4.127 (Thermo Electron Corporation, Dreieich, Germany).

| Raman spectroscopy at 785 nm
A B&W TEK Inc. i-Raman® Plus system was used with a laser wavelength of 785 nm and a BWS465-785S spectrometer using a scan range of 174-3200 cm −1 ; the resolution was <4.5 cm −1 at 912 nm. A BAC151B Raman Video Microsampling System was applied with an objective lens magnification of 20× (camera: active pixels 1280 × 1024). The applied software was BWSpec® 4.10_4.
Integration time was chosen to reach a relative intensity above 45000 arbitrary units for the most intensive peak. Additional information on parameters can be found in Supporting Information S15 and S16. Integration time was chosen to reach a relative intensity above 45000 arbitrary units for the most intensive peak. Additional information on parameters can be found in Supporting Information S17 and S18.

| NMR method
Approx. 1 mg of the isolated unknown substance was dissolved in CDCl 3 and acetone-d 6 . The NMR spectra were recorded at room temperature with an AVANCE III HD 500 spectrometer and a 5 mm BBO Prodigy Cryo probe from BRUKER BioSpin (Karlsruhe, Germany).
Chemical shifts are reported in ppm relative to TMS ( 1 H/ 13 C: d = 0.00) as reference. The compound was fully characterized by 1D-and 2D-NMR spectra. The following spectra were recorded: 1D-1 H (single pulse experiment with 90 pulse, four scans, relaxation delay 63 s, exponential multiplication with line broadening 0.2 Hz), 1D-13 C (2000 scans, exponential multiplication with line broadening 1.0 Hz), 1  The spectra evaluation was performed by using the software MNova V. 14.1 Suite Expert (Mestrelab, Santiago de Compostela, Spain). The integrals of the 1D-1 H spectrum were defined by the line fitting mode "qGSD." The 1 H and 13 C spectra predictions were performed by using the MNova "NMR Predict" plugin with the "Modgraph NMRPredict Desktop" model with "fast increments" algorithm. The model has been additionally trained by the implementation of approx. 1500 own, fully assigned spectra of drugs and related substances.  While the partial structure of cumyl-1H-indole-3-carboxamide was already well known from other SCRAs, the cyclobutyl methyl group had to be examined more closely.

| RESULTS AND DISCUSSION
The position of the methylene group could be clearly determined due to the typical chemical shifts and correlation signals in the HMBC spectrum to the indole core and the aliphatic ring. For the less trivial determination of the cyclobutyl ring, the combined evaluation of information from all 1D and 2D NMR spectra was necessary. In the 1 H spectrum, for example, the signal of the methine group C22 splits into a septet, which clearly indicates the six neighboring protons. Four of them are assigned to the CH 2 signal from C23 and C25, whereas the other two protons belong to the C21 signal.
The aromatic signal group at 7.25 ppm is overlaid by the solvent signal. This signal pattern can be observed in the spectrum of the sample dissolved in acetone-d 6 (Supporting Information S6). The acetone spectrum was also used for the comparison with spectra of similar structures (Supporting Information S7). The acetone spectra do not provide full separation of the signals in the cyclobutyl ring. For that reason, the full structure assignment provided here are primarily on the basis of the chloroform spectra.
In the 13 C spectrum, the two isochronous (symmetric) carbon atoms C23 and C25 result in a single signal with approximately twice the intensity of the remaining methylene group of the ring (C24).
The correlation signals of the 2D spectra (Supporting Information S5 and S8-S10) also clearly prove the constitution of the indole ring system. Following the approach of the established semisystematic nomenclature used for synthetic cannabinoids, we proposed the name Cumyl-CBMICA following its identification (Cumyl-CycloButyl-Methyl-IndoleCarboxAmide). Table 1 shows the NMR assignments and correlations within H,H-COSY and HMBC spectra. Furthermore, we provide all measured data obtained during structure elucidation: the H,H-COSY NMR spectrum (Supporting Information S8), the H,C-HSQC NMR spectrum (Supporting Information S9), and the H,N-HMBC NMR spectrum (Supporting Information S10).

Analysis of a test-purchased herbal blend laced with
Cumyl-CBMICA by IR spectroscopy was complicated by the coextracted constituents from the plant matrix. Albeit, extraction of the active ingredient using an organic solvent like acetone or CHCl 3 , and subsequent acquisition of neat film ATR-IR spectra can be sufficient to generate a spectrum for the identification of the substance contained in the herbal matrix, if impurities greater than approx. 10% are present, serious interferences can be present and hamper the F I G U R E 2 A, gas chromatographyelectrospray ionization-mass spectrometry (GC-EI-MS) spectrum including proposed fragmentation of the new compound. B, liquid chromatography-quadrupole time of flight-mass spectrometry (LC-qToF-MS) spectrum. Measured and calculated m/z, suggested ion formulas of the fragments and mass errors are given in Supporting Information S20 T A B L E 1 Peak list for the assignments of signals from Cumyl-CBMICA including the structure of Cumyl-CBMICA, the COSY (blue), and HMBC (green) couplings  Note: Predicted values for δ were generated using the MNova "NMR predict" plugin of the software MNova V. 14.1.
successful identification. The protocol utilized here is steered towards reducing the presence of compounds from the plant matrix in the solution. In addition, the hyphenated method of a gas chromatograph interfaced with an IR detection system was used due to its capability to elucidate molecular structures and differentiate isomers in complex mixtures. The IR spectrum of a herbal blend extract recorded after chromatographic separation is shown in Figure 3. In GC-sIR, the compound is deposited cryogenically onto a ZnSe disc. The cryogenic deposition of the eluting compound occurs rapidly which could decrease the chance to form these hydrogen bonds. The deposited analyte is in the free base form and is believed to be of a partially amorphous crystal structure. The resulting GC-sIR spectrum is in good agreement with the solid ATR-IR spectrum. Because spectra acquired using GC-sIR are always from compounds in the free base form, the congruence of both spectra leads to the conclusion that the solid ATR-IR spectrum is also generated from the free base form of the compound.
The comparison of the GC-sIR and IR solid spectra reveals similarities but also some dissimilarities in the absorption bands. We suspect this is due to crystalline structures forming in the process of condensation after GC separation. In contrast, the solid isolated substance Overall, GC-sIR provides a powerful analytical method for the identification of analytes in mixtures or on herbal matrices. The detection method IR is considered an A-method in analytical chemistry for the unambiguous identification of a compound and differentiation from its isomers.
Additionally, Raman spectra at wavelengths of 785 nm (Supporting Information S15 and S16) and 1064 nm (Supporting Information S17 and S18) were recorded. In the case of the Raman spectra, the Raman shifts and an enlarged area of the Raman shifts are shown.
The spectra can also be used for the rapid identification of substances.