Structure-Activity Relationship of N-Ethyl-Hexedrone Analogues: Role of the α-Carbon Side-Chain Length in the Mechanism of Action, Cytotoxicity, and Behavioral Effects in Mice

Abstract

Synthetic cathinones are β-keto amphetamine derivatives whose appearance has increased dramatically in the past decades. N-Ethyl substituted cathinones have been proven to potently inhibit dopamine (DA) uptake and induce psychostimulant and rewarding effects in mice. However, little is known about the influence of the alpha-carbon side-chain length of N-ethyl cathinones on their pharmacological and toxicological effects. Thus, the aim of this study was to synthesize and investigate the in vitro and in vivo effects of five N-ethyl substituted cathinones: N-ethyl-cathinone (NEC), N-ethyl-buphedrone (NEB), N-ethyl-pentedrone, N-ethyl-hexedrone (NEH), and N-ethyl-heptedrone. HEK293 cells expressing the human DA or serotonin transporter (hDAT and hSERT) were used for uptake inhibition and binding assays. PC12 cells were used for the cytotoxicity assays. Swiss CD-1 mice were used to study the in vivo psychostimulant, anxiogenic, and rewarding properties. Our results show that all tested cathinones are able to inhibit DA uptake and are DAT-selective. The potency of DA uptake inhibitors increases with the elongation of the aliphatic side chain from methyl to propyl and decreases when increasing from butyl to pentyl, which correlates with an inverted U-shape psychostimulant response in mice at the medium dose tested. On the other hand, an increase in the α-carbon side-chain length correlates with an increase in the cytotoxic properties in PC12 cells, probably due to better membrane penetration. Moreover, all the cathinones tested have shown higher cytotoxicity than methamphetamine. Finally, our study not only demonstrated the rewarding properties of NEC and NEB but also the anxiety-like behavior induced at high doses by all the cathinones tested.

Keywords: anxiety; cytotoxicity; new psychoactive substances; psychostimulant; reward; synthetic cathinones.

Figure 1

Chemical structure of NEC, NEB, NEPD, NEH, and NEHP.

Figure 2

Competition binding curves of NEC, NEB, NEPD, NEH, NEHP, α-PVP, and cocaine on [³H]MPP⁺ uptake at DAT and [³H]5-HT uptake at SERT (panel A,B) and [³H]WIN35,428 binding at DAT and [³H]Imipramine binding at SERT (panel C,D) in transfected HEK293 cells. Data are expressed as a percentage of control uptake (mean ± SEM) of four independent experiments carried out in triplicate.

Figure 3

Evaluation of the cytotoxicity potential of synthetic cathinones in NGF-differentiated PC12 cells using the WST-8 assay. Results are expressed as a percentage (%) of cell viability (mean ± SEM) of 3–4 experiments carried out on triplicates. Tukey’s multiple-comparison test: *p < 0.05, **p < 0.01, and ***p < 0.001 vs the corresponding control (0 mM) group, #p < 0.05 and ###p < 0.001 vs the matching concentration of Meth.

Figure 4

Effects of NEC (panel A), NEB (panel B), NEPD (panel C), NEH (panel D), and NEHP (panel E) on cumulative HLA in mice. Tukey’s multiple-comparison test: *p < 0.05, **p < 0.01, and ***p < 0.001 vs saline, ###p < 0.001 vs 3 mg/kg, $$p < 0.01, and $$$p < 0.001 vs 10 mg/kg. Panel F represents the effects of the NECs, α-PVP, and cocaine at 10 mg/kg on cumulative HLA in mice. Tukey’s multiple-comparison test: *p < 0.05, **p < 0.01, and ***p < 0.001, ##p < 0.01 and ###p < 0.001 vs α-PVP, $p < 0.05 and $$$p < 0.001 vs cocaine. Bars represent mean ± SEM of the total distance (cm) traveled in 60 min. N = 11–14/group.

Figure 5

Time course profile of HLA induced by NEC (panel A), NEB (panel B), NEPD (panel C), NEH (panel D), and NEHP (panel E). Each time point represents the mean ± SEM of the distance (in cm) traveled in 5 min blocks. Only comparisons vs the corresponding saline group are shown for clarity purposes. Tukey’s multiple-comparison test: *p < 0.05, **p < 0.01 and ***p < 0.001, 3 mg/kg vs saline, #p < 0.05, ##p < 0.01, and ###p < 0.001 10 mg/kg vs saline, $$p < 0.01 and $$$p < 0.001 30 mg/kg vs saline. N = 13–14/group.

Figure 6

Effects of NEC (A), NEB (B), NEPD (C), NEH (D), and NEHP (E) on the OF test (anxiety-like behavior) in CD-1 mice. Bars represent mean ± SEM of time in the center, expressed as a percentage vs its corresponding saline. Tukey’s multiple-comparison test: *p < 0.05 and **p < 0.01 vs saline. N = 10/group.

Figure 7

Effects of NEC (panel A), NEB (panel B), NEPD (panel C), NEH (panel D), NEHP (panel E), and α-PVP and cocaine (panel F) on the CPP test in mice. Bars represent the mean ± SEM of the preference score (difference between the time spent in the drug-paired compartment on the test day and the preconditioning day). Tukey’s multiple-comparison test: **p < 0.01 and ***p < 0.001 vs saline. N = 12–14/group.

Scheme 1. Synthesis of N-Ethyl Cathinones