New psychoactive substances, compounds structurally similar to already legislatively controlled illicit drugs recently appearing on the drug scene with increasing frequency, pose an increasing global problem.
The EU Early Warning System for NPS currently monitors over 670 substances, with 51 reported in 2017 for the first time and 101 in 2014. In addition, the total number of NPS all over the world moni- tored by the United Nations Office on Drugs and Crime is 803 individual NPS. In 2016, about 71,000 seizures of NPS were reported through the EU Early Warning and almost one- third of these seizures are of synthetic cathinones. New psychoactive substances are often considered by users as a safe alternative to illicit narcotics and psychotropic substances, despite the fact that they possess similar struc- tures and psychoactive effects. Structural modification of the parent NPS may often lead to the enhancement of hallucino- genic or psychostimulative effects, e.g.
in case of synthetic cannabinoids and cathinones. Synthetic cathinones are the sec- ond most frequently intercepted NPS. Their effects include empathy, openness, increased energy and libido of consum- ers, but also dangerous neurological, cardiac and psychiatric signs and symptoms.
Methylone and pentylone studied here are homologues in the class of synthetic cathi- nones. Unlike natural cathinone, methylone and pentylone both possess the methylenedioxy functional group on the phenyl ring, similarly to the well-known psychoactive sub- stance 3,4-methylenedioxyamphetamine, better known as ecstasy.
Due to this structural similarity, methy- lone and pentylone combine the effects of cathinone and MDMA.
Their mechanism of action is based primarily on inhibiting the uptake of norepinephrine, dopamine and serotonin by monoamine neurotransmitter transporters.
However, the full metabolism in the human body is not yet completely understood.
Methylone and pentylone differ in the length of the α-alkyl chain; methylone contains a methyl group and pen- tylone a propyl group.
According to pharmacokinetic studies on rats, a longer α-alkyl chain causes higher lipophi- licity, hence, pentylone exhibits a higher plasma concentra- tion than methylone after dosing the same amount of the pure substance.
However, safety data concerning their toxic- ity on humans as well as information about the side effects of long-term usage are unavailable.
The current trends in the control of NPS demand a fast and reliable analysis.
Moreover, methods for a detailed description of their structure are required.
However, most studies are focused on the detection of NPS in bio- logical samples performed by high-performance liquid chromatography or gas chromatography with mass spectrometry. For studies on identification of real NPS samples, vibrational spectroscopy techniques such as infrared or Raman spectroscopy are often used.
The advantage of this approach is a fast and reliable analysis, which can even be performed by portable spectrometers. Molecular spectroscopy is one of the effective tools for the identification of the NPS. Since synthetic cathinones are chiral, chiroptical methods can be used for their detailed study. The chiroptical methods, specifically vibrational cir- cular dichroism, electronic circular dichroism and Raman optical activity, in combination with theoretical quantum chemical predictions can provide fur- ther 3D structural information on NPS in solution, which is crucial, e.g.
for the understanding of their biological activity and toxicity. The comparative analysis of experimental chiroptical properties and DFT calculations also allow the determination of the absolute configuration of individual separated enantiomers. Analyses per- formed by different independent chiroptical spectroscopic methods provide more reliable structural characterizations of the studied molecules. The precise structural characterization of enantiomers, enabled by the combination of chiroptical spectroscopic methods and DFT calculations, may be helpful for under- standing differences in their toxicity. These methods can also be used to determine the presence of a particular drug enantiomer in a mixture of different compounds—a typi- cal feature of synthetic cathinone products available on the Internet. There are many examples that enantiomers of licit as well as illicit drugs have different properties and tox- icity. Apart from the widely known Contergan affair, it has already been shown in literature that enantiomers of some widely used drugs of abuse, e.g.
ketamine and deschlo- roketamine possess significantly different properties in terms of pharmacological effect and toxicity.
With the increasing number of synthetic cathinones, the need for detailed information on the structure of individual enantiom- ers and structure-properties relationships is essential. This is due to the fact that naturally occurring active substance is-cathinone, and it has already been documented that- enantiomers of synthetic cathinones are in many cases more potent/toxic than-enantiomers.
It is therefore evident that not only simple identification of a substance by analytical methods, but also more in depth structural information is required, and in this case, DFT calculations represent an important tool.
In the continuation of our systematic study of NPS, we introduce a comparison of two NPS homologues— methylone and pentylone—in an aqueous solution.
Employ- ing the methods of VCD, ECD, IR spectroscopy and ultra- violet absorption spectroscopy supported by the DFT calculations at B3LYP/6-311++G or B3PW91/6 311++G levels, including solvent effects, we elucidated in detail the 3D structure of both drugs and compared their structural features. Moreover, we performed chiral separa- tion of the studied substances and determined the absolute configuration of the respective enantiomers.
Racemic standards of methylone Deuterium oxide for the dissolution of methylone and pentylone was purchased from ISOSAR GmbH, Germany.
HPLC grade solvents were obtained from Labicom and ethanol was purchased from Merck. Diethylamine, used as a basic additive, was obtained from Sigma-Aldrich.
Enantioseparation Both the analytical and the preparative chiral separation of the drugs were performed using the Waters AutoPurification System equipped with a UV–Vis detec- tor.
The analytical separa- tion was performed on a chiral polysaccharide column Chi- ralArt Amylose-SA with an analyte concentration of 1 mg ml −1 and a flow rate of 1 ml min −1 at ambient tem- perature. For the preparative chromatography, an analogous polysaccharide column available in our laboratory—Chiral- pak IA—was used.
For methylone, the sample con- centration was 17.5 mg ml −1 , while in case of pentylone, for which a different mobile phase was required, the sample concentration was 4 mg ml −1 .
In both cases, at flow rate of 15 ml min −1 and ambient temperature conditions were employed.
For analytical chromatography screening, com- mercially available drugs were dissolved in the mobile phase containing 0.1% of diethylamine. The sample for preparative chromatography was dissolved in a mixture of 1% diethyl- amine in propan-2-ol and a total volume of 1 ml was adjusted by the addition of heptane. In both cases, the addition of diethylamine ensured the liberation of a free base of the drug from the corresponding hydrochloric acid salt. Electronic circular dichroism The experimental ECD spectra were acquired using a J-815 spectrometer purged with nitrogen gas during the measurement.
To measure the ECD spectra, solutions of individual enantiomers of a concen- tration of 15 mg l −1 in demineralized water were prepared.
These sample solutions were pipetted into a quartz cuvette with a pathlength of 1 cm and measured in a spectral range of 185–380 nm at ambient temperature with a 20 nm min −1 scanning speed, 8-s response time, 3 accu- mulations, and 1 nm band width. The baseline was corrected by subtracting the spectra of demineralized water obtained under identical experimental conditions. The corresponding UV absorption was calculated from the detector HT voltage using the Spectra Analysis module of the Spectra Manager software. The VCD and IR absorption spectra were acquired using the FTIR IFS 66/S spectrometer equipped with a VCD/IRRAS PMA 37 module, a ZnSe beam splitter, a BaF 2 polarizer, and an MCT detector.
The solutions of individual enantiomers of methylone hydrochloride and pentylone hydrochloride in D 2 O were placed individually into a BioCell cuvette with CaF 2 windows and an optical pathlength of 27.3 μm.
The spectra were measured at ambient temperature in a spectral range of 1750 to 1250 cm −1 with a resolution of 8 cm −1 . The VCD spectra were averaged from 9 to 12 blocks, each measured for 20 min and containing 3680 interferometric records. The baseline of each enantiomer was corrected by the subtrac- tion of the solvent spectra measured under identical experimental conditions. The VCD noise spectra were offset from zero for clarity. Quantum chemical calculations The starting geometries of-methylone and-pentylone hydrochlorides were optimized using the DFT method at the B3LYP/6-31G level. Reoptimization of the final lowest energy conformers, considering their Gibbs free energies, was performed by the DFT method at several higher levels of theory, B3PW91/aug-cc-pVDZ, B3PW91/6-311++G, CAM-B3LYP/6-311++G, CAM-B3LYP/aug-cc-pVDZ, wB97XD/6-311++G, wB97XD/aug-cc-pVDZ, and wB97XD/TZVP, Electronic Supplementary Material, Table S1 and S2) using the Gauss- ian 09 program package. In this work, only the results providing the best level of agreement with the experimen- tal spectra are presented in detail, B3PW91/6-311++G).
All the calculations were carried out on a supercomputer Altix UV 2000. In general, 12 processors with total memory of 64 GB were used for individual computation tasks, which took 40–48 h. The D 2 O solvation effect was considered via the con- ductor-like polarizable continuum model. The DFT calculations of VCD spectra included the effect of deuteration of the amine hydrogens.
The population weighted average VCD, ECD, IR and UV spectra were calculated using the Boltzmann distribution based on Gibbs free energy at a temperature of 298 K. The similar- ity overlaps of the experimental and calculated spectra were undertaken using the CDSpecTech program, based on the similarity of the dissymmetry factor to determine quantitatively the agreement between both spectra and to choose the optimal scaling factor.
A scaling factor of 1.0 marks the maximum similarity.
For visualization of the simulated VCD and IR spectra, a Lorentz profile function with a 10 cm −1 bandwidth was assumed.