The social problems related to the uncontrolled use of psychoactive compounds, including new synthetic cannabinoids, motivate researchers to learn about their properties and to develop methods for their identification. MDMB-4en-PINACA is an example of an illegally traded synthetic cannabinoid that requires precise detection procedures. Some fractions of analytical data concerning this compound can be found in the literature [11,12,13,14,15,16]. They are consistent with those we obtained using some basic analytical techniques, such as GC–EI-MS, FTIR spectroscopy, LC–MS and 1H NMR spectroscopy. The present paper reports additional information from product ion spectra at different CEs, 2D NMR, UV–VIS spectroscopy and Powder X-ray diffraction measurements that significantly broadens the analytical knowledge of MDMB-4en-PINACA.
The obtained sample of MDMB-4en-PINACA had well-developed crystal line. The material was used for the powder X-ray diffraction. Unfortunately, the sample did not contain monocrystals suitable for the single crystal diffraction. Attempts for the recrystallization did not result in the growth of monocrystals, and it was decided to conduct only powder diffraction measurements. For the acquired powder diffractogram (Fig. S8), the parameters of elementary cell were determined, but the determination of crystal lattice structure was not possible. Although the crystallographic data for synthetic cannabinoids are scarce, it can be seen that most of these compounds form crystals of monoclinic system with density in the range of 1.1–1.4 g cm−3 [17, 24]. Analogous structural features were observed for the MDBM-4en-PINACA sample. For the powder diffraction measurements of organic compounds, majority of the peaks appear in the 2θ range below 50°. In the case of MDMB-4en-PINACA, most of the significant signals were observed within the mentioned region, with the most intense and characteristic peaks placed below 20°.
Most of MDMB-4en-PINACA proton signals were clearly assigned to the corresponding nuclei. The differentiation of the overlapping aromatic and pent-4-en-1-yl chain resonances was conducted using heteronuclear 2D techniques. These could be done due to the greater dispersion of 13C signals for mentioned fragments leading to the well-separated correlation peaks. This was especially useful for 7.38–7.42 ppm signals corresponding to proton H6 and H7 (Table 2, Fig. 1).
The acquired 2D NOESY spectrum contained multiple cross-peaks (see Table 2) corresponding to the through-space correlations between protons. In the case of tert-leucinate and indazole groups, observed nuclear Overhauser effect (NOE) signals led to the connectivities similar to those from other 2D spectra. The only novel conformational information was obtained from cross-peaks of pent-4-en-1-yl protons. Signals correlating nuclei H1a, H2a and H3a to the aromatic protons of 7.38–7.42 ppm (Table 2) indicated the presence of small intramolecular distances between considered atoms pairs. Although the 7.38–7.42 ppm region contained overlapping signals from protons H6 and H7, the NOE typically occurs, when the internuclei distance does not exceed 4.5 Å, which excludes the possibility of NOE for pairs of H6-aliphatic proton. Hence, the aromatic—pent-4-en-1-yl cross-peaks were assigned to the correlations H7-H1a, H7-H2a and H7-H3a (see Table 2). Considering the possible conformations of MDMB-4en-PINACA aliphatic chain, it could be noticed that protons H2a and H3a may have differing positions and distances with respect to nuclei H7 in particular rotamers. The steric hindrance between considered aliphatic protons may lead to the conformations with H2a being in close proximity to aromatic proton, and H3a with distance to H7 greater than 4 Å, and opposite situation with H3a oriented near H7 nucleus. The former conformation would lead to the NOE cross-peak occurring only between H7 and H2a, while the latter would give correlation signal for pair H7-H3a. As those correlations were observed in 2D NOESY, it indicates that MDMB-4en-PINACA rotamers occurring in solution contain both of the described H2a and H3a orientations. Moreover, very weak cross-peaks between vinyl and aromatic protons were also observed indicating long intramolecular distances between nuclei or small abundance of rotamers with these structural feature.
Conformational analysis of MDMB-4en-PINACA
Although multitude of physicochemical techniques can be employed for structural characterization of synthetic cannabinoids, only the single crystal X-ray diffraction is able to determine precisely the atom positions and the resulting conformations. The other way is to perform the geometry optimization of the molecule by quantum chemical methods. The results obtained using B3LYP DFT  functional for MDMB-4en-PINACA indicate the presence of five stable conformers of the molecule (see Fig. S9 in supplementary material). For each of the obtained rotamers, the conformation of carboxamide moiety is fixed by intramolecular hydrogen bonding. The amide group, which is one of the synthetic cannabinoid parts responsible for the bonding to the cannabinoidical receptors, should have a substituent generating steric hindrance required for effective binding . The position of the tert-leucinate group, a steric substituent in MDMB-4en-PINACA, does not change during conformational transitions. The only fragment of MDMB-4en-PINACA with conformational flexibility is the pentenyl chain. Considering the structures of the obtained rotamers and their populations, none of the determined chain orientations is highly favored over others (see abundances of rotamers at Fig. S9 caption). Although the presence of the alkene double bond may suggest the possibility of π–π stacking with the indazole ring, such interaction did not occur in the stable conformers. This fact is also confirmed by the experimental results. In the case of π–π stacking, the centers of interacting groups are in close proximity, which leads to multiple NOE signals between the protons of the interacting groups. 2D NOESY spectrum showed only a very small correlation signal of protons 5a and 7 (see Table 2, Fig. 1), which is consistent with the edge-to-edge orientation of the vinyl and indazole ring in conformers a and b (see Fig. S9). The lack of π–π interactions is related to the polarized electrostatic nature of the indazole ring. The nitrogen atoms of the indazole and carbonyl group attached to the ring cause a significant polarization of aromatic moiety leading to the change in its quadrupole moment. A significant negative partial charge, being a component of the quadrupole moment, is distributed above both aromatic ring faces in the aromatic rings without electron-withrawing motifs . For the indazole part of MDMB-4en-PINACA, the negative partial charge is distributed in the regions close to the nitrogen atoms. On the other hand, in the subsequent region of the heteroaromatic group, the deficit of electron density is observed. This distribution of electrostatic potential favors π–π interactions to the π fragments with electron realizing groups. No such effect was observed in MDMB-4en-PINACA due to fact that only a simple vinyl group is present in the described analyte.
GC–MS and ATR-FTIR are most frequently used in routine identification of synthetic cannabinoids, including MDMB-4en-PINACA. In these cases, the substance identification is based on the comparison of EI mass spectra or infrared spectra with those in the database (e.g., NIST database or SWGDRUG mass spectral library). Although it ensures fast routine workflow, a more elaborate analysis of the EI and IR spectra should also be conducted. It is especially apparent for the vibrational spectra of carboxyamideindazole with regions below 1600 cm−1 being the place of multiple signals (Fig. S10). Most of the bands appearing in these range cannot be simply assigned with the “rule of thumb” as little is known about the character of their normal modes, e.g. for MDMB-4en-PINACA. For this compound, it is difficult to differentiate between the bands of indazole and vinyl vibrations and those of alkyl and carbonyl fragments in the range of low wavenumbers. The theoretical vibrational analysis of MDMB-4en-PINACA allowed us to assign the positions of the multiple bands to the stretching and the out-of-plane bending modes of the indazole ring in the regions of 1600–1300 cm−1 and of 1000–700 cm−1, respectively. Yet, the indazole normal modes are influenced by the vibrations of the rest of the molecule. The normal modes of the ring stretching (1600–1300 cm−1) involve the aromatic C–H bond rocking. Moreover, a small contribution of C–(C=O)–N bending is observed in the out-of-plane bending modes (1000–700 cm−1). For the vinyl group, the out-of-plane vibrations were assigned to the signals in 1007 cm−1 and 925 cm−1 positions. Contrary to the enlisted modes of indazole, the normal modes of the vinyl group include insignificant contributions from other parts of the molecule.