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

Abstract

We herein 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. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucosedeprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1′Z)-1,1′-(1,3phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC 50 = 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. Bis-nitrones are well-known antioxidant and neuroprotective agents showing high clinical potential. For instance, bis-nitrone W-AZN (Fig. 1), an azulenyl spin trap possessing neuroprotective effects in an animal model of

Synthesis, Neuroprotection, and Antioxidant Activity of 1,1′-Biphenylnitrones as α-Phenyl-N-tert-butylnitrone Analogues in In Vitro Ischemia Models

“…To this end, we used the XTT cell viability test and a colorimetric assay that detects the cellular metabolic activities. Based on a previous work from our laboratory [ 22 ], we selected the appropriate experimental conditions and tested the neuroprotective effect of BPNs 1-5 at different concentrations (0.1–1000 μM), which were added 10 min before the administration of O10 μM /R30 μM (O/R), and using PBN and NAC as reference compounds, at the same concentrations (0.1–1000 μM), as reference compounds [ 22 , 25 ].…”

“…Next, the neuroprotective effect of BPNs 1–5 was evaluated in an in vitro oxygen glucose deprivation (OGD) model, followed by oxygen and glucose resupply (R), and an in vitro model of ischemia reperfusion (IR) [ 22 , 26 ]. Tested compound concentrations ranged from 0.01 to 1000 μM, after IR.…”

“…Assessment of cell viability. Measurements of cell viability in human SHSY5Y neuroblastoma cells were carried out in 96-well culture plates as described [ 22 , 26 ]. Briefly, control and treated SH-SY5Y neuroblastoma cells (about 0.75–1×105 cells/well) were incubated with the XTT solution (Cell Proliferation Kit II (XTT), Sigma, Aldrich, Madrid) at 0.3 mg/mL final concentration for 2 h in a humidified incubator at 37 °C with 5% CO 2 and 95% air (v/v), and the soluble orange formazan dye formed was spectrophotometrically quantified, using a Biotek Power- Wave XS spectrophotometer microplate-reader at 450 nm (reference 650 nm).…”

“…For these assays, cultured neuroblastoma cells grown in 96-well culture dishes at a density of 1.5 × 105 cells/well were used. LDH activity was measured as the rate of decrease of the absorbance at 340 nm, resulting from the oxidation of NADH to NAD+ as described [ 22 , 28 ]. Data are given as the percentage of LDH release with respect to the total LDH content (LDH in the culture medium and LDH inside the cells).…”

“…Bis-nitrone TN-2 ( Figure 1 ) bears two tert -butyl nitrone motifs anchored to a pyrazine as the heterocyclic ring core, showing high neuroprotective effect in in vitro and in vivo models of stroke, as a very plausible consequence of its ability to trap ROS HO ● and O 2 ●- [ 21 ]. In this context, we have recently identified (1 Z ,1′ Z )-1,1′-(1,3-phenylene)bis( N -benzylmethanimine oxide) ( HBN6 ) ( Figure 1 ) as a potent neuroprotective ligand overcoming the neuroprotective and antioxidant capacities of PBN [ 22 ].…”