Xavier Berzosa

Background/Objectives: Synthetic cathinones are a rapidly evolving class of new psychoactive substances whose structural diversity complicates toxicological risk assessment. Methylenedioxy cathinones occupy a pharmacological space between MDMA-like entactogens and more dopaminergic stimulant cathinones, but their direct cardiac liabilities remain poorly characterized. This study aimed to compare the cardiotoxic and neurobehavioral profiles of methylone, butylone, pentylone and their N,N-dimethyl analogues, and to determine how alkyl-chain extension and terminal amine substitution shape functional toxicity. Methods: Wild-type short-fin zebrafish (Danio rerio) embryos were used as a multiparametric New Approach Methodology. Cardiac rhythmicity was assessed in 3 days post-fertilization embryos after acute exposure to methylone, butylone, pentylone, dimethylone, dibutylone, dipentylone, dihexylone and diheptylone by high-speed video microscopy and dynamic pixel-based analysis, focusing on atrial chronotropy and atrioventricular conduction. Basal locomotor activity was evaluated in 5 days post-fertilization eleutheroembryos over 120 min using automated video tracking. Results: Negative chronotropy increased with alkyl-chain extension, with the monoalkyl subset following the rank order methylone < butylone < pentylone. Among dialkyl analogues, dihexylone and, especially, diheptylone produced the strongest atrial-rate inhibition. AV conduction impairment was more heterogeneous but became prominent among higher-liability analogues, with diheptylone showing the lowest AV-block midpoint descriptor and complete lethality at 1000 µM. Locomotor profiling revealed predominantly hypoactive phenotypes, with sustained late-phase inhibition especially for dipentylone, dihexylone and diheptylone. Conclusions: Alkyl-chain extension and terminal amine substitution shaped cardiac and neurobehavioral toxicity in a structure-dependent manner. The zebrafish workflow provides a structure-oriented framework for prioritizing emerging methylenedioxy cathinones with comparatively higher functional cardiac liability.

Sartans are an important family of drugs used to treat hypertension. A common characteristic of most of them is a biphenyl moiety, commonly obtained through a Suzuki-Miyaura cross-coupling reaction, of which thousands of tons are produced every year. Bayesian Optimization (BO), a machine-learning technique to find the optimum of complex unknown functions with a minimum number of test points, has been applied to optimize the reaction conditions in continuous flow with a focus on lowering the costs and increasing both the productivity and the sustainability of the process, as evaluated through its Process Mass Intensity (PMI). Although this is a common chemical transformation, the fact that it happens in a biphasic medium with organometallic catalysis makes its optimization more challenging. Six variables were identified as important for the reaction system: residence time, temperature, equivalents of the nonlimiting reactant, mol % of catalyst, mol % of ligand, and solvent volumes. With less than 40 experiments, the BO algorithm achieved solutions that increased the productivity up to 4 times compared to the initial results of the DOE experiments and reduced the cost by around 30%. A Pareto front with different optimal solutions was provided at the end of the optimization, and the point with the lowest cost and the lowest PMI was scaled up to increase productivity while keeping the unitary cost unchanged.

Synthetic cathinones are a class of new psychoactive substances (NPS) whose structural diversity and rapid evolution complicate toxicological risk assessment. Methylenedioxy cathinones, including methylone, butylone, pentylone and their N,N-dimethyl analogues, occupy a pharmacological space between MDMA-like entactogens and more dopaminergic stimulant cathinones. Although their monoamine transporter profiles and psychostimulant effects have been partially characterized, their direct cardiac liabilities remain poorly understood. Here, we used zebrafish embryos as a multiparametric New Approach Methodology to compare the cardiotoxic and neurobehavioral profiles of methylone, butylone, pentylone, dimethylone, dibutylone, dipentylone, dihexylone and diheptylone. Cardiac rhythmicity was assessed after acute exposure by high-speed video microscopy and dynamic pixel-based analysis, focusing on atrial chronotropy and atrioventricular (AV) conduction. Negative chronotropy increased with alkyl-chain extension, with the monoalkyl subset following the rank order methylone < butylone < pentylone. Among dialkyl analogues, dihexylone and especially diheptylone produced the strongest atrial rate inhibition. AV conduction impairment was more heterogeneous but became prominent among the higher-liability analogues, with diheptylone showing the lowest AV-block EC50 and complete lethality at 1000 µM. Time-resolved locomotor profiling revealed predominantly hypoactive phenotypes, with the most sustained late-phase inhibition observed for dipentylone, dihexylone and diheptylone. Integration of locomotor and cardiac endpoints showed that locomotor effects generally occurred below concentrations producing severe AV conduction impairment, although endpoint separation varied across analogues. Overall, this study provides a structure-oriented zebrafish framework for prioritizing emerging methylenedioxy cathinones with comparatively higher functional cardiac liability.

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.

The rapid emergence of New Psychoactive Substances (NPS), particularly pyrrolidinophenone derivatives, poses a significant challenge for public health and forensic toxicology. While their neuropharmacological profiles as dopamine transporter inhibitors are well-documented, their cardiac toxicity remains poorly understood. This study employs a multiparametric New Approach Methodology (NAM) using zebrafish embryos to integrate neurobehavioral and cardiotoxic endpoints for comparative hazard prioritization. We evaluated nine pyrrolidine-containing cathinones, including α-PVP, MDPV, α-PiHP, MDPiHP, α-D2PV, 3-Cl-, 4-Cl-, and 3,4-Cl-α-PVP, and 4-F-3-Me-α-PVP, on locomotor activity and cardiac rhythmicity using high-speed video microscopy and dynamic pixel analysis. Across the series, compounds induced concentration-dependent negative chronotropy and, in most cases, locomotor suppression. Crucially, we identified a functional dissociation between atrial rate control and atrioventricular (AV) conduction. The 3,4-dichloro substitution (3,4-Cl-α-PVP) was the most potent inducer of negative chronotropy (EC₅₀ = 52.6 μM), whereas 4-Cl-α-PVP exhibited a distinct pro-arrhythmic liability, increasing the incidence of 2:1 AV block. Time-course locomotor profiling indicated that α-PVP and chlorinated analogs were among the most potent behavioral modifiers. Using a Functional Safety Index (AV block EC₅₀/locomotor EC₅₀-like), we show that most compounds exhibit wide separations between neurobehavioral inhibition and severe conduction impairment, while specific substitutions, particularly para-chlorination, are associated with comparatively reduced functional separation between these endpoints within the assay. Overall, these data demonstrate that subtle structural changes within the pyrrolidinophenone scaffold can shape distinct arrhythmic phenotypes and functional safety profiles, supporting zebrafish-based integrated screening as a rapid platform for prioritizing emerging synthetic cathinones with comparatively higher cardiac liability within this experimental framework.