Synthesis, antioxidant properties and neuroprotection of \u03b1-phenyl-tert-butylnitrone derived HomoBisNitrones in in vitro and in vivo ischemia models

Beatriz Chamorro,Daniel Diez-Iriepa,José Marco-Contelles,David García-Vieira,Francisco López-Muñoz,María Jesús Oset-Gasque,Isabel Iriepa,Mourad Chioua,Ricardo Martínez-Murillo,Dimitra Hadjipavlou-Litina,Belén Merás-Sáiz,Israel Fernández,Daniel Gonzàlez-Nieto

doi:10.1038/s41598-020-70690-y

Abstract

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs1–9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs1–9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1′Z)-1,1′-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 μM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.

Highlights

We report the synthesis, antioxidant power and neuroprotective properties of nine homo-bisnitrones HBNs 1–9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy
One of the first events taking place in the initial stages of stroke is the collapse of the mitochondrial electron transport chain (ETC), which leads to extended cell death and brain damage due to the formation of reactive oxygen species (ROS)
In order to mimic this event into suitable experiments, we tested the effect of the bisnitrones on cell death induced by Oligomycin A and Rotenone (O/R), inhibitors of mitochondrial complexes V and I, respectively

Summary

Introduction

We report the synthesis, antioxidant power and neuroprotective properties of nine homo-bisnitrones HBNs 1–9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. Homo-bis-nitrones (HBNs) 1–9 result from the incorporation of a second identical nitrone moiety at the para (p-HBNs 1–3), meta (m-HBNs 4–6) and ortho (o-HBNs 7–9) positions and bearing methyl, tert-butyl or benzyl substituents, respectively, as the N-alkyl groups attached to the nitrone motif Among these nitrones, only HBNs 121, 222, 323, 424,25, and 526 have been previously described in the literature, but in studies not related to their antioxidant properties and/or potential use for stroke therapy. The hypothesis behind the present design is that two “nitrone” scavenging motifs in the same scaffold should afford a higher antioxidant power than only one As it will be shown later on, we have identified (1Z,1′Z)-1,1′-(1,3phenylene)bis(N-benzylmethanimine oxide) (HBN6) as a potent neuroprotective ligand (EC50 = 1.24 ± 0.39 μM), whose neuroprotective and antioxidant capacities are higher than those of PBN

Methods

Results

Conclusion