Synthesis, characterization and monoamine transporter activity of the new psychoactive substance mexedrone and its N-methoxy positional isomer, N-methoxymephedrone

Abstract

3-Methoxy-2-(methylamino)-1-(4-methylphenyl)propan-1-one (mexedrone) appeared in 2015 and was advertised by UK Internet retailers as a non-controlled mephedrone derivative (2-(methylamino)-1-(4-methylphenyl)propan-1-one), which was of particular interest to countries who operate generic drugs legislation. This study describes the synthesis and analytical characterization of mexedrone and the differentiation from its isomer, N-methoxymephedrone, which was predicted to be a suitable candidate before the identity of mexedrone was revealed. A full analytical characterization is described using various chromatographic, spectroscopic and mass spectrometric platforms and X-ray crystal structure analysis. The analytical data obtained for a vendor sample were consistent with the synthesized mexedrone reference standard and analytical differentiation between the mexedrone and N-methoxymephedrone positional isomers was achieved. Furthermore, α-chloromethylmephedrone was identified as a by-product during mexedrone synthesis. All three substances were also studied for their uptake and releasing properties at dopamine transporters (DAT), norepinephrine transporters (NET) and serotonin transporters (SERT) using in vitro monoamine transporter assays in rat brain synaptosomes and compared to mephedrone. Mexedrone was a weak non-selective uptake blocker with IC₅₀ values in the low μM range. It was also devoid of releasing activity at DAT and NET but displayed weak releasing activity at SERT (EC₅₀ = 2.5 μM). The isomer N-methoxymephedrone was found to be a weak uptake blocker at DAT, NET and SERT, as well as a fully efficacious substrate-type releasing agent across all three transporters with EC₅₀ values in the low micromolar range. The synthesis by-product α-chloromethylmephedrone was inactive in all assays. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: chemistry; mephedrone; mexedrone; new psychoactive substances; psychostimulants.

Figure 1

(A) Chemical structures of mephedrone, mexedrone and isomeric N-methoxymephedrone. (B and C) Synthesis pathways employed for mexedrone and N-methoxymephedrone. (D) Synthesis pathway employed for the route specific chlorinated by-product.

Figure 2

Gas chromatography mass spectrometry data. (A and B) Electron ionization mass spectra for mexedrone and N-methoxymephedrone. (C) Proposed fragments for both isomers.

Figure 3

(A and B) Product ion spectra of synthesized mexedrone and N-methoxymephedrone obtained from in-source collision induced dissociation at increased fragmentor voltage (110 V). Insert: Partial HPLC ESI single quadrupole MS trace to illustrate the separation of both isomers.

Figure 4

The suggested dissociation pathways for mexedrone (A) and N-methoxymephedrone (B) with HR-MS providing elemental compositions with acceptable mass accuracies consistent with the proposed structures.

Figure 5

Molecular structure recorded for the mexedrone vendor sample using X-ray crystal structure analysis.

Figure 6

Effects of mexedrone, N-methoxymephedrone, α-chloromethylmephedrone and comparison test drug mephedrone on inhibition of uptake and stimulation of release at DAT, NET and SERT in rat brain synaptosomes. For uptake assays (A, B and C), synaptosomes were incubated with different concentrations of the test drugs in the presence of 5 nM [³H]dopamine (A, for DAT), 10 nM [³H]norepinephrine (B, for NET) or 5 nM [³H]serotonin (C, for SERT). Data are expressed as percentage of [³H]transmitter uptake (mean± SD) for n =3 experiments. For release assays (D, E and F), synaptosomes were preloaded with 9 nM [³H]MPP⁺ for DAT (D) and NET (E) or 5 nM [³H]serotonin for SERT (F), then incubated with different concentrations of test drugs to evoke release via reverse transport. Data are expressed as percentage of [³H]substrate release (mean ± SD) for n = 3 experiments.