Structure-activity relationships of bath salt components: substituted cathinones and benzofurans at biogenic amine transporters

Abstract

Rationale: New psychoactive substances (NPSs), including substituted cathinones and other stimulants, are synthesized, sold on the Internet, and ingested without knowledge of their pharmacological activity and/or toxicity. In vitro pharmacology plays a role in therapeutic drug development, drug-protein in silico interaction modeling, and drug scheduling.

Objectives: The goal of this research was to determine mechanisms of action that may indicate NPS abuse liability.

Methods: Affinities to displace the radioligand [¹²⁵I]RTI-55 and potencies to inhibit [³H]neurotransmitter uptake for 22 cathinones, 6 benzofurans and another stimulant were characterized using human embryonic kidney cells stably expressing recombinant human transporters for dopamine, norepinephrine, or serotonin (hDAT, hNET, or hSERT, respectively). Selected compounds were tested for potencies and efficacies at inducing [³H]neurotransmitter release via the transporters. Computational modeling was conducted to explain plausible molecular interactions established by NPS and transporters.

Results: Most α-pyrrolidinophenones had high hDAT potencies and selectivities in uptake assays, with hDAT/hSERT uptake selectivity ratios of 83-360. Other substituted cathinones varied in their potencies and selectivities, with N-ethyl-hexedrone and N-ethyl-pentylone having highest hDAT potencies and N-propyl-pentedrone having highest hDAT selectivity. 4-Cl-ethcathinone and 3,4-methylenedioxy-N-propylcathinone had higher hSERT selectivity. Benzofurans generally had low hDAT selectivity, especially 1-(2,3-dihydrobenzofuran-5-yl)-N-methylpropan-2-amine, with 25-fold higher hSERT potency. Consistent with this selectivity, the benzofurans were releasers at hSERT. Modeling indicated key amino acids in the transporters' binding pockets that influence drug affinities.

Conclusions: The α-pyrrolidinophenones, with high hDAT selectivity, have high abuse potential. Lower hDAT selectivity among benzofurans suggests similarity to methylenedioxymethamphetamine, entactogens with lower stimulant activity.

Keywords: Benzofurans; Cathinones; New psychoactive substances; Pharmacology; Psychostimulant; Transporter.

Figure 1.

Structures of substituted cathinones, benzofurans, prolintane, METH and MDMA at physiological pH.

Figure 2.

Concentration-response curves of 12 substituted cathinones for inhibition of [³H]DA uptake by hDAT, [³H]5-HT uptake by hSERT and [³H]NE uptake by hNET. (A) 4-F-α-PVP, (B), 4-Cl-α-PVP, (C) TH-PVP, (D) 3,4-MDPHP, (E) 4-CPD, (F) α-EPP, (G) N-propyl PD, (H) 4-MEAP, (I) MCAT, (J) 3-MMC, (K) 4-CEC and (L)Mexedrone. Data are the mean and S.E.M. of three or more experiments, each conducted in duplicate.

Figure 3.

Concentration-response curves for [³H]neurotransmitter release by METH, MCAT, and 7 substituted cathinones and benzofurans. (A) MCAT, (B) 4-CEC, (C) Mexedrone, (D) 2-MAPB, (E) 5-MAPB, (F) 6-MAPB, (G) 5-MAPDB, (H) 6-APB and (I) METH. The AUC for each drug concentration was normalized to the maximal METH effect. hDAT: [³H]DA release. hSERT: [³H]5-HT release. hNET: [³H]NE release. Data are the mean and S.E.M. of three or more experiments, except when a drug had no releasing efficacy, when data are the mean and range of two experiments.

Figure 4.

Alignment of all ligands in the binding pocket. A. Ligand alignment based on the paroxetine binding pose derived from hSERT crystal structure (PDB: 5I6X). Key pharmacophore features such as positive charge, aromatic ring and hydrophobic are shown in the dotted lines. B. Slab view of hSERT and the central site showing aligned ligands. C. An enlarged view of central site and plausible ligand binding poses, dotted lines indicate subsites A, B, and C.

Figure 5.

Modeling of representative ligands. A) Superimposition of predicted docking poses of TH-PVP against the three transporters, hSERT residues are labeled and indicated with orange stick and carton, hDAT and hNET are represented in green and pink colors, respectively. The sequence comparison table in the bottom shows aligned sequences of the transporters in the same position. The color code is the same for all the panels except for panel C. B) Predicted pose of 4-Cl-α-PVP. C) Overlay of the predicted binding poses 4-Cl-α-PVP (blue stick) and TH-PVP (pink stick), and protein in orange. The selectivity is caused by the different position of the butyl tail of each ligand. For clarity, only hSERT is shown. D) Pentedrone E) N-propyl PD F) 5-MAPB.