AbstractSince the global COVID-19 pandemic began, the scientific community has dedicated efforts to finding effective antiviral drugs to treat or minimize the effects caused by the SARS-CoV-2 coronavirus. Some targets can act as inhibitor substrates, highlighting the Main Protease (Mpro), which plays an essential role in the translation and transcription of the virus cycle. Withanolides, a class of natural C28steroidal lactones, are compounds of interest as possible inhibitors of Mproand other critical targets of the virus, such as papain-like protease. In this study, the isolation of a new withanolide (1), along with the known 27-deoxywithaferin A (2) and 27-deoxy-2,3-dihydrowithaferin A (3), from the leaves of Athenaea velutina(Solanaceae) is described. Their structures were determined using spectroscopic and spectrometric methods (NMR, IR, HRESIMS). Moreover, the interaction and the stability of withanolides 1–3and withanolide D (4), previously isolated of Acnistus arborescens, against the Mprotarget through molecular docking, molecular dynamics, and binding free energy simulations were analyzed. The molecular dynamics results indicated that the complexes formed by the molecular docking simulations between the Mprotarget with each of the withanolides 1–4exhibited good stability during the simulations due to a slight change in the structure of complexes. The binding free energy results suggested that withanolide (1) can be a natural candidate against COVID-19 disease.Communicated by Ramaswamy H. Sarma