Abstract
<jats:p>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 &lt; butylone &lt; 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.</jats:p>