N10 -carbonyl-substituted phenothiazines inhibiting lipid peroxidation and associated nitric oxide consumption powerfully protect brain tissue against oxidative stress

Abstract

During some investigations into the mechanism of nitric oxide consumption by brain preparations, several potent inhibitors of this process were identified. Subsequent tests revealed the compounds act by inhibiting lipid peroxidation, a trigger for a form of regulated cell death known as ferroptosis. A quantitative structure-activity study together with XED (eXtended Electron Distributions) field analysis allowed a qualitative understanding of the structure-activity relationships. A representative compound N-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-10H-phenothiazine-10-carboxamide (DT-PTZ-C) was able to inhibit completely oxidative damage brought about by two different procedures in organotypic hippocampal slice cultures, displaying a 30- to 100-fold higher potency than the standard vitamin E analogue, Trolox or edaravone. The compounds are novel, small, drug-like molecules of potential therapeutic use in neurodegenerative disorders and other conditions associated with oxidative stress.

Keywords: XED; antioxidant; ferroptosis; lipid peroxidation; neurodegeneration; nitric oxide; phenothiazine.

Figure 1

Representative traces (a) and (b) and summary of the data (c) of NO consumption after addition of the NO donor DETA/NO (200 μM) to buffer or cerebellar cells (20 × 10⁶ cells/ml; 1.25 mg protein/ml) in the absence or presence of compound numbers 26, 27, 11, 19, 15, and 91. Data are n = 2 ± SD

Figure 2

DT‐PTZ‐C inhibits iron/ascorbate toxicity with greater potency than Trolox. (a) Representative photomicrographs of PI‐stained hippocampal slices 24 hr following exposure to ascorbate (500 μM) and increasing concentrations of FeSO₄ (0–1 mM). (b) Summary data (mean ± SEM; n = 8 slices) are expressed as percentage death in the three major hippocampal regions (CA1, CA3, and dentate gyrus). (c) The concentration‐dependent effects of DT‐PTZ‐C and Trolox were assessed against maximum slice toxicity (500 μM ascorbate + 1 mM iron) and are expressed as mean ± SEM % death in CA1, n = 8 slices

Figure 3

DT‐PTZ‐C inhibits ABAP toxicity with greater potency than Trolox. (a) Representative photomicrographs of hippocampal slices stained with PI 24 h following exposure to ABAP (0.3–3 mM). (b) Summary data (mean ± SEM; n = 8 slices) are expressed as percentage death in CA1. (c) The concentration‐dependent effects of DT‐PTZ‐C and Trolox were assessed against maximum slice toxicity (3 mM ABAP) and are expressed as mean ± SEM % death in CA1, n = 8 slices

Figure 4

DT‐PTZ‐C inhibits NO consumption with a similar potency to aromatic imines. Summary data (mean ± SEM; n = 4) of hemoglobin‐coated bead absorbance following incubation for 25 min with pellet (0.1 mg/ml), supernatant (10%), and DETA/NO (100 μM). Increasing concentrations (0.3–1,000 nM) of (a) compound 26, (b) phenothiazine, (c) iminostilbene, and (d) phenoxazine were included to determine their potency at inhibiting NO consumption

Figure 5

(a) QSAR model of antioxidant activity using calculated descriptors. (b). Details of the model. (c) DT‐PTZ‐C field analysis using Cresset. Negative field points—blue; positive field points—dark red; van der Waals surface field points—yellow, hydrophobic field points—gold/orange. (d) Field analysis of a weakly active compound 41

Figure 6

Comparison of IC₅₀'s for phenothiazine‐based compounds in the hemoglobin bead versus TBARS assays. (a) A significant correlation between the two assays was found (Pearson's rank correlation coefficient, r = 0.92)

Figure 7

Lipid peroxidation activity of phenothiazines proceeds via generation of a radical cation or potentially a radical (drawn as oxygen centered). Stabilization of the structures is proposed to occur via the tautomeric form