Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure–activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear “hybrid” properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.

Changes in the molecular structure of synthetic cathinones has led to an increase in the number of novel emerging drugs in the illicit drug market at an unprecedented rate. Unfortunately, little is known about the neuropsychopharmacology of recently emerged halogen-substituted α-PVP derivatives. Thus, the aim of this study was to investigate the role of para- and meta-halogen (F-, Cl-, and Br-) substitutions on the in vitro, in silico, and in vivo effects of α-pyrrolidinopentiophenone (α-PVP) derivatives. HEK293 cells expressing the human dopamine or serotonin transporter (hDAT and hSERT) were used for the uptake inhibition and transporter affinity assays. Molecular docking was used to model the interaction mechanism against DAT. Swiss CD-1 mice were used for the horizontal locomotor activity, open field test, and conditioned place preference paradigm. All compounds demonstrated potent DA uptake inhibition and higher DAT selectivity than cocaine. Meta-substituted cathinones showed higher DAT/SERT ratios than their para- analogs, which correlates with an increased psychostimulant effect in vivo and with different meta- and para-in silico interactions at DAT. Moreover, all compounds induced rewarding and acute anxiogenic effects in mice. In conclusion, the present study demonstrates the role of meta- and para-halogen substitutions in the mechanism of action and provides the first evidence of the rewarding and anxiety-like properties of halogenated α-PVP derivatives.

Synthetic cathinones are emerging psychoactive substances designed to mimic the effects of classical psychostimulants. Among them, α-D2PV, a novel pyrrolidine-containing cathinone characterized by a phenyl group on the α-carbon atom, has gained significant attention. This study investigates the in vitro and in silico mechanism of action as well as the abuse liability of α-D2PV using rodent models. Dopamine (DA), noradrenaline (NE), and serotonin (5-HT) uptake inhibition assays were conducted in HEK293 cells expressing the corresponding human monoamine transporter, complemented by molecular docking studies at the DA transporter (DAT). Behavioral studies in male Swiss CD-1 mice assessed locomotor activity and conditioned place preference, while microdialysis and self-administration experiments were performed in male Sprague-Dawley rats. The findings reveal that α-D2PV is a potent DA and NE uptake inhibitor, with minimal activity at the 5-HT transporter (SERT). Docking studies showed that the benzene rings of α-PVP and α-D2PV align precisely in their most stable conformations at DAT. In vivo, α-D2PV elicited dose-dependent hyperlocomotion, thigmotaxis, and rewarding effects in mice, alongside increased extracellular DA levels in the nucleus accumbens of awake rats. Self-administration experiments confirmed α-D2PV's high reinforcing efficacy, indicating a significant risk of abuse in humans. Finally, these results underscore the necessity for continued surveillance of α-D2PV within the illicit drug market. Furthermore, novel synthetic cathinones incorporating a phenyl ring at the α-carbon side chain warrant proactive monitoring due to their potential to retain dopaminergic activity and evade initial legal controls.

N‐ethyl‐pentylone (NEP), also known as ‘ephylone’ and N‐ethylnorpentylone, has been identified as one of the most recent novel psychostimulants to emerge into the illicit drug market and it has been associated with some intoxications and even fatalities. However, little is known about the consequences of its repeated consumption as well as the role of the monoaminergic system in such consequences. Thus, the aim of our study was to investigate the neurochemical profile and the behavioural effects after both acute and repeated NEP exposure. Male OF1 mice were acutely (1, 3, 10 mg/kg, i.p.) or repeatedly (1, 3, 10 mg/kg, i.p., 5 days, twice/day) exposed to NEP, and anxiety‐like behaviour, aggressiveness, social interaction, depressive‐like symptoms, body temperature, changes in monoaminergic enzymes and neurotransmitters levels as well as ΔFosB in striatum and prefrontal cortex (PFC) from post‐mortem tissue were analysed short after drug‐exposure or during drug‐withdrawal. Acute administration of NEP induced anxiolytic effects but also an aggressive behaviour and social exploration deficits in mice, which persist during NEP‐withdrawal. Moreover, NEP induced hyperthermia as well as depressive‐like symptoms after repeated administrations that may be related to the decrease in serotonin and noradrenaline levels observed in striatum and PFC. Finally, the long‐term increase in ΔFosB levels in striatum after NEP chronic exposure points to a high risk of dependence. Altogether indicates that NEP consumption induces different neurological and neuropsychiatric disorders accompanied by changes in the monoaminergic system, posing a threat to public health. image