Synthesis , absolute configuration and in vitro cytotoxicity of deschloroketamine enantiomers : Rediscovered and abused dissociative anesthetic

In this study, we aim to determine differences in cytotoxicity of racemic deschloroketamine and its enantiomers. The synthesized racemate of this recently rediscovered and abused dissociative anaesthetic was resolved by chiral HPLC and the absolute configuration of the enantiomers was assigned using a combination of circular dichroism methods and single-crystal X-ray. Not only the absolute configuration, but also the most preferred conformers present in the crystal were successfully determined by electron and vibrational circular dichroism supported by ab initio calculations, and confirmed by X-ray. The in vitro cytotoxicity of racemic deschloroketamine and its enantiomers was determined for nine different types of cell lines. Generally, (S)-deschloroketamine exhibited higher cytotoxicity in the majority of cases. For human embryonic kidney cells (HEK 293T), the (S)-enantiomer reached the IC50 below 1 mM concentration and, in consequence, proved to be twice as potent as the (R)-enantiomer. On the other hand, live-cell fluorescence microscopy imaging at sub-IC50 concentrations provided evidence for only a minor effect of deschloroketamine racemate and enantiomers on endoplasmic reticulum stress and mitochondria morphology in neuroblastoma cells SH-SY5Y.


Enantioseparation of racemic deschloroketamine
Deschloroketamine was originally proposed for clinical use in the racemic form, therefore, there is no information on its enantioseparation available in the patent literature.Recent studies focused on its characterization also neglected the fact that the substance exists in two enantiomeric forms.Thus, some estimations of the chromatographic behavior of the substance had to be performed before proceeding with the enantioseparation.Generally, deschloroketamine is -ketoamine, thus, it has a similar structure to cathinonesanother type of new psychoactive substances.There are several reports on chiral resolution of cathinones on various types of chiral stationary phases ranging from polysaccharide over crown ethers to chiral ion exchangers.S1-3 The highest efficiency in the enantioseparation of new chiral substances is usually achieved with polysaccharide chiral columns.Therefore, we decided to use amylose-based column, which is available in preparative size in our laboratory and optimize the chromatographic conditions for this chiral column.Racemic DCK hydrochloride subjected to enantioseparation was well soluble only in low molecular mass alcohols.However, the enantiomer separation under such polar organic mode conditions on analytical amylose-based column (ChiralArt Amylose-SA) was not efficient.Since much higher success rate of enantioseparation on polysaccharide chiral stationary phases is generally achieved in normal mobile phase mode (mixture of alkanes with alcohols), S4 we decided to transform DCK hydrochloride into a free base.The samples were prepared in the following way: DCK hydrochloride (10 mg) was suspended in propan-2-ol (500 l), diethylamine (10 l) was added and the suspension was shaken until it cleared.Then, a mixture of heptane/propan-2-ol (9/1) was added, the achieved solution was filtered through a syringe filter (0.43 m pores) and used for DCK resolution.For the preparative method development, we used a screening technique proposed by the column manufacturer and started with hexane/propan-2-ol (9/1) mixture.First, mobile phase without any additives was employed.No enantiomer resolution was achieved with this first choice mobile phase.Other tested mobile phase compositions with hexane as the bulk component and propan-2-ol or ethanol as polar additives were also not successful.Thus, we changed hexane for heptane, as sometimes, there may be a slight change in enantioselectivity connected with higher density of heptane.Indeed, with a mobile phase consisting of heptane/propan-2-ol (9/1) partial resolution of DCK enantiomers was achieved.To reduce non-enantioselective interactions of the basic analyte with the acidic silica support, diethylamine (0.1% in mobile phase) was introduced.This change led to further improvement of peak resolution (Figure S1a).Reducing the amount of propan-2-ol (Figure S1b) and finally to 5%, acceptable base-line resolution was achieved.The optimum preparative method (Figure S1c) was used for the enantioseparation of DCK.

Ab initio calculations
The differences between the two major conformers of model A can be characterized by the value of the dihedral angle α (Figure S2 and Table 1).As it can be seen from the results in Table I, both levels of the theory used in calculations provided similar results.Table 1.
The lowest-energy conformers of DCK, their relative free energies, Boltzmann populations and dihedral angle describing relative orientation of -NH 2 CH 3 group for the model A.

ECD spectroscopy
The assignment of absolute configuration obtained from experimental VCD data and in silico simulation of VCD spectra was further reinforced by the match between experimental and in silico predicted ECD spectra.In this case, also the major orbital contributions to the experimentally observed ECD bands were visualized (Figure S4).

Figure S4.
Major orbital contributions to experimentally observed ECD bands.

Figure S2 .
Figure S2.Definition of the dihedral angle α which describes orientation of the -+ NH 2 CH 3 group.
1 H spectrum of ketone 7 13 C spectrum of ketone 7 HSQC spectrum of ketone 7 1 H spectrum of bromoketone 8 13 C spectrum of bromoketone 8 HSQC spectrum of bromoketone 8