Since methylone and pentylone are in the cathinone family, they can be enantiomerically resolved by several different methods including, for example, capillary electrophoresis, supercritical fluid chromatography, and HPLC. However, only some methods from this list can provide pure enantiomers in preparative amounts of the milligram to gram scale. The most prominent method for the separa- tion of chiral substances nowadays is preparative HPLC. We have already employed this method in our previous study focused on the determination of the absolute con- figuration of butylone, thus HPLC was selected as the method of choice in this study. Starting from the previously optimized conditions, we have found that although they are in the same family of NPS, methylone, butylone and pentylone exhibit signifi- cantly different chromatographic behaviours. Therefore, different methods of chiral separation for methylone and pentylone have been developed; more detailed information on the screening of the mobile phase conditions can be found in the Electronic Supplementary Material.
Meth- ylone, as the more polar substance, was resolved using a mobile phase composed of a mixture of hexane/EtOH with diethylamine, whereas the more lipophilic pentylone was successfully resolved using a mixture of heptane/IPA with diethylamine.
In both cases, automatic fraction collection was set to ensure high purity of the collected enantiomers. In order to assure the stability of the separated enantiomers and to avoid a cyclization reaction occurring for the cathinone free base, the separated substances were transformed to hydrochlorides by adding an etheric solution of hydro- gen chloride.
Both enantiomers were found to be of comparable optical purity with the enantiomeric excess of > 96.3%.
This value was still acceptable for the subsequent spectroscopic measure- ments.
For pentylone, even better values of > 99.4% and > 98.9% for the first and the second eluting enantiomers, respectively, were found.
Conformational analysis The individual-enantiomers of methylone and penty- lone hydrochloride were selected for the conformational analysis. Energetically preferred geometries of these mol- ecules were determined by three or five dihedral angles α 1 , α 2 , α 3 , and optionally α 4 and α 5 for methylone and pentylone, respectively. The dihedral angle α 1 was systemati- cally changed by 180° rotation about a single bond and the remaining angles α 2 , α 3 and possibly α 4 and α 5 by 120° using the MCM software. As a result, 18 and 162 starting geometries for methylone and pentylone, respectively, were obtained. Those geometries were subsequently optimized by the DFT method at the B3LYP/6-31G level of theory. Since most starting structures were converging to the same geometry or their population was too low, conformational analysis revealed all the relevant stable conformers: 5 and 9 for methylone and pentyl- one, respectively. These stable conformers were reoptimized at a more refined level of theory: B3LYP/6-311++G or B3PW91/6-311 ++G to provide the final structures and their relative abundances based on the Boltz- mann distribution.
All the conformers were stabilized by the hydrogen bond- ing between the oxygen atom of the carbonyl group and the hydrogen atom of the amino group. From all the conformers of methylone, this interaction most stabilized the structure of the conformer M I, probably resulting in its high relative abundance.
The distance of hydrogen and oxygen in the case of the conformer M I was ~ 2.1 Å, for other con- formers of methylone this distance was 2.2–2.5 Å. As for the individual dihedral angles, the angle α 1 occupied predominantly two positions that differed by ~ 180°.
The total number of stable conformers of pentylone was higher than methylone due to the elongation of the alkyl chain and the associated increase in the number of dihedral angles in the molecule. All 9 conformers of pentylone were also sta- bilized by the hydrogen bonding between the oxygen of the carbonyl group and the hydrogen of the amino group, with the distance of these atoms being in the range of 2.0–2.2 Å for all the stable conformers. Relatively small differences in the length of this stabilizing interaction may be the reason or the more uniform relative abundances of conformers than in the case of methylone. Electronic spectroscopy Since the experimental ECD spectra of the first and the sec- ond eluting compounds were mirror images, the enantiomeric character of the separated products was con- firmed. The comparison of the experimental and simulated spectra determined the absolute configuration of the indi- vidual enantiomers: methylone 1 and 2 were determined as- and-enantiomer, respectively. On the contrary, pen- tylone 1 and 2 were determined as- and-enantiomer, respectively.
These results could be confirmed further using the similarity overlap plots.
For the correct assignment of the bands in the experimental and simulated spectra, the simu- lated spectra of methylone were scaled by a factor of 0.96 in order to reach the maximum value of the similarity index. After scaling, a similarity index of 0.85 was achieved in this case.
For pentylone, a scaling factor of 0.97 was applied and the corresponding index of similarity reached 0.75. Both results were more than sufficient to reliably determine the absolute configuration of individual enantiomers, which was subsequently confirmed by a detailed VCD spectra analysis.
In the experimental ECD spectra of methylone and penty- lone hydrochloride, six very similar bands reflecting the combination of electron transitions π → π *, n → π * and n → σ * were observed. The bands at 330 nm and partially at 278 nm were mainly an expression of an electron transition from the highest occupied to the lowest unoccupied molecu- lar orbitals. Three positive and three negative bands in the spectra of-enantiomers were also present in the simulated weighted average spectra of-enantiomers. The spectra of individual conformers of the-enantiomers differed considerably from one another, indicating the exceptional sensitivity of ECD to the 3D structure of the molecules compared to non-polarized methods. However, the experimental ECD spectra did not allow reliable differentiation between the two studied compounds. The simulated UV spectra of-methylone and- pentylone hydrochloride correctly predicted four significant bands of the experimental spectra. The simulated spectra were scaled by a factor of 0.97 and 0.98 for methy- lone and pentylone, respectively, and the final similarity overlap reached 0.82 and 0.78 for methylone and pentylone, respectively. However, this method does not seem to be suit- able either for the differentiation of substances differing in the number of –CH 2 groups nor for the studies of the 3D structural changes of the individ- ual conformers.
The shapes of the partial spectra were very similar, with slight differences up to 10 nm observed in the change of the position of the band maximum at 330 nm. Differences between the intensities of band at ~ 234 nm in the simulated spectra of-pentylone were caused mainly by the change of dihedral angle α 1 : if its value was positive, the intensity of the band was lower.