Structure–activity relationship of synthetic cathinones: integrated in silico, in vitro, and in vivo studies of α-PiHP analogues

Synthetic cathinones represent the main group of emerging psychostimulants consumed worldwide. Structural modifications of these compounds have led to pyrovalerones, potent dopamine transporter (DAT) inhibitors associated with high abuse liability. Among them, α-pyrrolidinoisohexanophenone (α-PiHP) became the most seized pyrovalerone in the European Union in 2023. Novel analogues have rapidly appeared, yet their neuropharmacological and toxicological properties remain unknown. This study aimed to characterize α-PiHP and five structurally related derivatives (3-Me-α-PiHP, 4-Me-α-PiHP, 3-F-α-PiHP, 4-F-α-PiHP, and MDPiHP). Monoamine transporter activity was evaluated using HEK293 cells expressing human norepinephrine (hNET), hDAT, or serotonin (hSERT) transporters through uptake inhibition and binding assays. Molecular docking was performed to model interactions at hDAT. Psychostimulant, thigmotactic, and rewarding effects were evaluated in male Swiss CD-1 mice using horizontal locomotor activity, open field, and conditioned place preference paradigms. Lethality after drug administration was also assessed. All compounds potently inhibited hDAT and hNET, while displaying limited activity at hSERT. This resulted in high hDAT/hSERT ratios indicative of a stimulant-like profile and elevated abuse liability, confirmed in vivo by pronounced psychostimulant and rewarding effects. All molecules interacted with the orthosteric binding site at hDAT and displayed higher binding affinity than cocaine in in vitro and in silico studies. Meta-substituted analogues showed greater hDAT affinity and behavioural responses than para-isomers. Notably, MDPiHP exhibited the highest potency and affinity, strongest psychostimulant effects and significant lethality. These findings demonstrate that minor structural modifications markedly influence pharmacological activity of pyrovalerone derivatives, highlighting the high abuse potential and health risks associated with emerging α-PiHP analogues.