[1,2,5]Oxadiazolo[3,4- b]pyrazine-5,6-diamine Derivatives as Mitochondrial Uncouplers for the Potential Treatment of Nonalcoholic Steatohepatitis

Abstract

Small molecule mitochondrial uncouplers are emerging as a new class of molecules for the treatment of nonalcoholic steatohepatitis. We utilized BAM15, a potent protonophore that uncouples the mitochondria without depolarizing the plasma membrane, as a lead compound for structure-activity profiling. Using oxygen consumption rate as an assay for determining uncoupling activity, changes on the 5- and 6-position of the oxadiazolopyrazine core were introduced. Our studies suggest that unsymmetrical aniline derivatives bearing electron withdrawing groups are preferred compared to the symmetrical counterparts. In addition, alkyl substituents are not tolerated, and the N-H proton of the aniline ring is responsible for the protonophore activity. In particular, compound 10b had an EC₅₀ value of 190 nM in L6 myoblast cells. In an in vivo model of NASH, 10b decreased liver triglyceride levels and showed improvement in fibrosis, inflammation, and plasma ALT. Taken together, our studies indicate that mitochondrial uncouplers have potential for the treatment of NASH.

Figure 1.

Chemical structures of select protonophore mitochondrial uncouplers.

Figure 2.

Oxygen consumption rate comparing 10b to BAM15 in L6 myoblasts. BAM15 EC₅₀ = 0.486 ± 0.057 μM and Compound 10b EC₅₀ = 0.191 ± 0.014 μM. Data are averaged over 3 separate experiments. Error bars represent standard error of mean.

Figure 3.

Body temperature (A) was not altered by acute oral dose of up to 100 mg/kg 10b. Tissue distribution of 10b was determined 1 h after acute oral dose of 50 mg/kg body weight (B). n=5–6 male mice per group for body temperature (A) and n=3 male mice per group for tissue distribution (B). Statistical significance was assessed by Two-Way Repeated Measures ANOVA with Dunnett’s correction.

Figure 4.

Effect of compound 10b on liver histopathologic and biochemical phenotypes in the STAM mouse model. Representative images of Hematoxylin and Eosin staining and Sirius Red staining in liver sections (A), Sirius Red staining quantified as a measure of fibrosis (B). NAFLD Activity Score (C) is the sum of individual parameters: steatosis (D), inflammation (E), and ballooning (F). Graphs show mean ± SEM. * indicates p < 0.05, by Kruskal-Wallis test with Dunn’s correction for multiple comparisons for non-parametric data. n=8 male mice per group.

Figure 5.

Effect of compound 10b and telmisartan on body composition and biochemistry in the STAM mouse model. Telmisartan, but not 10b, significantly decreased body weight (A), liver weight (B) and liver-to-body weight ratio (C). Both 10b and telmisartan significantly decreased liver triglyceride (D), but only telmisartan affected plasma triglyceride (E) and cholesterol (F) Telmisartan, but not 10b, exacerbated hyperglycemia (G). Plasma ALT levels were reduced by both 10b and telmisartan (H) but plasma AST levels were unaffected (I). Graphs show mean ± SEM. * indicates p < 0.05, by One-Way ANOVA with Dunnett’s correction for multiple comparisons for normally distributed data (A, D,G–I) or Kruskal-Wallis test with Dunn’s correction for multiple comparisons for non-parametric data (B–C,E–F). n=8 male mice per group.

Scheme 1.

Synthesis of symmetrical (A) and unsymmetrical (B) derivatives. Reagents and conditions: (a) oxalic acid, 10% HCl, reflux, 3 h, quantitative; (b) PCl₅, POCl₃, reflux, 2 h, 30%; (c) aniline, THF, reflux, 19 h, 19 – 95%; (d) ortho-fluoroaniline, Et₃N, THF, 0 °C, 0.25 to 1 h; (e) amine, Et₃N, THF, 0 °C to rt, 19 h, 13 – 59%.