Eugenol-Derived Piperazine Mannich Bases as Selective Inhibitors of Mayaro Virus: In Vitro Antiviral Evaluation and Mechanistic Insights.

Arboviruses represent an ongoing global health challenge, particularly in tropical regions where diseases caused by Chikungunya virus (CHIKV) and Mayaro virus (MAYV) continue to emerge. Given the absence of licensed antiviral therapies, the identification of effective small molecules remains critical. This study evaluated sixteen eugenol- and dihydroeugenol-derived piperazine Mannich bases for antiviral activity against CHIKV, MAYV, and Zika virus (ZIKV) in Vero cells. Cytotoxicity was assessed using the MTT assay, and antiviral activity was quantified through median effective concentration (EC50) and selectivity index (SI) values. Time-dependent cytopathic effect inhibition, plaque reduction assays, virucidal assays and docking were performed to characterize the mechanism of action. Three compounds (D3, D7, and E7) exhibited selective antiviral activity. Notably, compound D7 demonstrated low-micromolar anti-MAYV inhibition (EC50 = 14.43 μM; SI > 16.6), surpassing ribavirin by more than thirty-fold in relative potency. Time-dependent assays revealed progressive viral titer reductions of up to 3 log10 PFU/mL over 48 h. Virucidal assays confirmed that D7 does not directly inactivate viral particles. These findings identify D7 as a promising antiviral lead compound and provide a foundation for further mechanistic, optimization, and in vivo studies aimed at developing anti-alphavirus therapeutics.