Oxadiazolopyridine Derivatives as Efficacious Mitochondrial Uncouplers in the Prevention of Diet-Induced Obesity

Abstract

Small-molecule mitochondrial uncouplers are gaining recognition as potential therapeutics for metabolic diseases such as obesity, diabetes, and nonalcoholic steatohepatitis (NASH). Specifically, heterocycles derived from BAM15, a potent and mitochondria-selective uncoupler, have yielded promising preclinical candidates that are efficacious in animal models of obesity and NASH. In this study, we report the structure-activity relationship studies of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives. Using oxygen consumption rate as a readout of mitochondrial uncoupling, we established 5-hydroxyoxadiazolopyridines as mild uncouplers. In particular, SHM115, which contains a pentafluoro aniline, had an EC₅₀ value of 17 μM and exhibited 75% oral bioavailability. SHM115 treatment increased the energy expenditure and lowered the body fat mass in two diet-induced obesity mouse models, including an obesity prevention model and an obesity reversal model. Taken together, our findings demonstrate the therapeutic potential of mild mitochondrial uncouplers for the prevention of diet-induced obesity.

Figure 1.

Chemical structures of select small-molecule mitochondrial uncouplers.

Figure 2.

Structure–activity relationship studies of BAM15 toward the current work. Consensus Log P calculated using Reaxys (https://www.reaxys.com/).

Figure 3.

SHM115 is a mild mitochondrial uncoupler in L6 cells. (A,B) SHM115 and BAM15-stimulated OCR in L6 myoblasts. Values are graphed as (A) dose–response and (B) time course. (C) Self-limit test, in which SHM115 (25 μM) or vehicle was added to L6 cells before adding BAM15 (10 μM), and antimycin A (AntA, 10 μM) plus rotenone (Rot, 1 μM) were added at the indicated time. n = 4 wells per condition from three separate experiments. (D) Mitochondrial stress test, in which L6 cells were sequentially treated with oligomycin (oligo, 1 μM), SHM115 or BAM15 (drug, 50 μM), and antimycin A (AntA, 10 μM) plus rotenone (Rot, 1 μM) at the indicated time. n = 3 wells per condition. Values are represented as mean ± SEM.

Figure 4.

Profile of compound SHM115. (A) C57BL/6 male mice received SHM115 by either oral gavage at 10 mg/kg (per oral, p.o. n = 3) or intravenous injection at 1 mg/kg (i.v., n = 4). Values are represented as mean ± SEM. (B) Physicochemical and pharmacokinetic profiles of compound SHM115.

Figure 5.

Compound SHM115 increases the energy expenditure in mice fed a HFD and a high-sugar WD. (A,B) Whole-body oxygen consumption measurements (A) over time and (B) as average 2 h before and 4 h after gavage. (C,D) Whole-body carbon dioxide production measurements (A) over time and (B) as average before and after gavage. (E,F) RER was calculated (E) over time and (F) as average before and after gavage. (G,H) Locomotor activity measurements (G) over time and (H) as average before and after gavage. Values are represented as mean ± SEM, n = 7. *Indicates p < 0.05 compared to WD, determined by two-way repeated measures one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post hoc test.

Figure 6.

Compound SHM115 (130 mg/kg/d) prevents diet-induced obesity. (A,B) BW measurements as (A) % of initial over time and (B) represented as grams changed during the study. (C,D) Fat mass measurements, represented (C) as % of BW over time and (D) as grams at the end of the study. (E–G) Lean mass (fat-free mass) measurements, represented (E) as % of initial, (F) as % of BW over time, and (G) as grams at the end of the study. Values are represented as mean ± SEM, n = 5–7. *Indicates p < 0.05 compared to WD, determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test for nonparametric data.

Figure 7.

130 mg/kg/d of compound SHM115 prevents WD-induced glucose intolerance. (A,B) Mice were administered a GTT both (A) pre- and (B) post-treatment. (C) Change in the AUC pre- and post-treatment. *Indicates p < 0.05 compared to WD, determined by one-way ANOVA, followed by Kruskal–Wallis test. (D) Fasting (5 h) and (E) random-fed blood glucose measurements pre- and post-treatment. (F) Random-fed plasma insulin levels pre- and post-treatment. Values are represented as mean ± SEM, n = 5–7. *Indicates p < 0.05 compared to WD, determined by two-way repeated measures ANOVA, followed by Dunnett’s multiple comparisons post hoc test. #Indicates p < 0.05 compared to pre-treatment measurements, determined by two-way repeated measures ANOVA, followed by Sidak’s multiple comparisons post hoc test. Values are represented as mean ± SEM, n = 5–7.

Figure 8.

Tissue distribution of compound SHM115. SHM115 concentration was measured (A) at the end of the prevention study (130 mg/kg as food admixture, n = 7) and (B) 2 h after oral administration at 10 mg/kg (n = 3). (C) Tissue distribution of SHS4121705 2 h after oral administration at 10 mg/kg (n = 3). Values are represented as mean ± SEM.

Figure 9.

Compound SHM115 (130 mg/kg/d) reverses diet-induced obesity without altering the food intake or lean mass. (A) BW measurements as % of initial and (B) average of food intake. (C,D) Fat and lean mass (fat-free mass) measurements, represented as % of initial at the end of the study. (E–I) Fat pads and muscle weights measured after 3 weeks of treatment. (J) Fecal triglycerides (TG) and (K) FFAs measured at day 11 of treatment. (L) Rectal temperature measured at day 11 of treatment. Values are represented as mean ± SEM, n = 5–8. *Indicates p < 0.05 compared to WD, determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test or Kruskal–Wallis test for nonparametric data.

Figure 10.

130 mg/kg/d of compound SHM115 improves glucose and insulin levels. (A,B) Mice were administered a GTT both (A) pre- and (B) post-treatment. (C) Change in the AUC pre- and post-treatment. (D,E) Fasting (5 h), (D) blood glucose, and (E) insulin measurements pre- and post-treatment. (F) Fasting plasma FFA levels pre- and post-treatment. Values are represented as mean ± SEM, n = 5–8. *Indicates p < 0.05 compared to WD, determined by two-way repeated measures ANOVA, followed by Dunnett’s multiple comparisons post hoc test. #Indicates p < 0.05 compared to pre-treatment measurements, determined by two-way repeated measures ANOVA, followed by Sidak’s multiple comparisons post hoc test.

Scheme 1.. Synthesis of Oxadiazolopyridine 7^a

^aReagents and conditions: (a) N-chlorosuccinimide, acetic acid, 100 °C, 2 h, and 85%; (b) PIDA, acetone, 80 °C, 16 h, and 80%; (c) PPh₃, THF, 0 °C to rt, 0.5 h, and 54%; (d) MeOH, NaH, THF, rt, 0.5 h, and 92%; (e) Pd₂(dba)₃, XantPhos, amine, K₂CO₃, dioxane, 85–110 °C, 16 h, and 15–88%; and (f) Na₂CO₃, 1:1 water/dioxane, 110 °C, 2–16 h, and 20–98%.