Small molecule mitochondrial uncouplers are compounds that dissipate the proton motive force independent of ATP synthase that results in increased energy expenditure. Mild mitochondrial uncoupling has therapeutic potential in treating obesity, diabetes, neurological diseases, non-alcoholic steatohepatitis (NASH), and aging. Our group has previously reported the discovery of a small molecule mitochondrial uncoupler BAM15, which was efficacious in an obesity mouse model. Herein, we describe the design and synthesis of two scaffolds as well as their characterization as mitochondrial uncouplers through a series of in vitro and in vivo assays. Compounds that pass as bona fide mitochondrial uncouplers are administered in mice to determine pharmacokinetic properties and promising compounds are then tested in a mouse model of obesity.
The first series of mitochondrial uncouplers are anilinopyrazines. By changing the substitution pattern and electronics on the aniline rings, our investigations reveal the importance of the proximity of aniline rings on the pyrazine core, with the 2,3-positions being crucial for uncoupling activity. We found that mitochondrial uncouplers 2.5g and 2.5l elicited a maximum oxygen consumption rate (OCR) of 260% and 343% with an EC50 of 2.5 and 5.9 µM, respectively. Utilizing the knowledge gained from the anilinopyrazine series, we designed a second novel chemical scaffold based on a related BAM15 analog 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol. The new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives have a pyridine instead of pyrazine core that is decorated with aniline substituents. We found that derivatives with electron withdrawing groups (EWG) substitutions in the 2,5-position on the aniline ring exhibited the greatest uncoupling activity compared to other structural isomers. Strong EWGs CF3/OCF3/SO2CF3 were well tolerated and demonstrated the highest uncoupling activity compared to other EWGs. Our studies indicated that placement of the hydroxyl group in the 2-position of the pyridine moiety was crucial for uncoupling activity. Several of the most promising compounds tested in vitro were examined in vivo and found to have good oral bioavailability in mice with ranges in Cmax of 10-90 µM and t1/2 of 0.9 to >24 hours. We found that analogs that have F/OCF3/SO2CF3 groups on the 4-position exhibited the longest t1/2 compared to other structural isomers, suggesting that this position is a site of metabolic lability. Among the 51 derivatives tested, SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR and an EC50 of 17.1 µM in L6 myoblast cells. SHM20519115 was found to have good oral bioavailability with a Cmax of 57 µM and a t1/2 of 4.4 hours. Additionally, SHM20519115 had significant distribution in adipose tissue where it can promote mitochondrial uncoupling. In a mouse model of obesity, SHM20519115 prevented fat mass gain by 59% compared to the western diet (WD) control group. Importantly, weight loss did not alter lean mass or food intake. Further characterization demonstrated that SHM20519115 prevented glucose and insulin intolerance in mice. Taken together, our investigations support the utility of mitochondrial uncouplers for the treatment of obesity and other metabolic disorders. / Doctor of Philosophy / Obesity is commonly considered a modern-day epidemic with more than 40% of adult Americans being classified as obese. The higher prevalence of obesity over the course of the last century has been attributed to a more sedentary lifestyle and high calorie diet. Obesity has been shown to negatively impact every organ system and increases the risk for heart disease, cancer, neurological diseases, non-alcoholic steatohepatitis (NASH), and diabetes. Moreover, obesity has further burdened the healthcare system with an estimated expenditure of $190 billion a year in the US. Although diet and exercise has shown excellent results in weight loss, long-term compliance with these regiments is extremely low. Current non-invasive treatments provide varying efficacies and a myriad of side-effects. Invasive procedures, which is restricted to those who are classified as 'morbidly obese' with a BMI > 40, have shown excellent results in facilitating weight loss but come with high cost and risks to patients. This excludes individuals in the BMI range of 30-40 unless they are qualified with additional comorbidities. In recent years, mitochondrial uncouplers have reemerged as a potential therapeutic treatment for obesity.
This dissertation discusses the structure-activity relationship study of anilinopyrazines and 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives as mitochondrial uncouplers. Building on previous work on BAM15, we investigated uncoupling activity of anilinopyrazines. We discovered that although anilinopyrazines were previously found to be inactive, modifications to the aniline rings could result in uncoupling activity. We found that strong electron withdrawing groups placed in the meta and para positions were most favorable. We also determined that the 2,3-disubstitution on the aniline rings was crucial for uncoupling activity. From this study, we discovered 2.5g and 2.5l that elicited a maximum oxygen consumption rate (OCR) of 260 and 343% with EC50 of 2.5 and 5.9 µM, respectively.
Furthermore, we recently reported a new series of 6-amino-[1,2,5]oxadiazolo[3,4-b]pyridin-5-ol derivatives and identified SHM20519115 as a mitochondrial uncoupler. Our studies determined that SHM20519115 demonstrated mild uncoupling activity with 48% BAM15 OCR with an EC50 of 17.1µM in L6 myoblasts cells. In a mouse model of obesity, SHM20519115 was found to be efficacious at a 130 mg/kg dose. Pharmacokinetic studies SHM20519115 showed greater overall distribution in adipose tissue in mice. Additionally, when examined in a mouse obesity prevention model, SHM20519115 successfully prevented 59% fat mass gain compared to the western diet (WD) control group. Finally, we found that SHM20519115 prevents glucose and insulin intolerance in mice. Taken together, these results support a role for mitochondrial uncouplers in the treatment of obesity.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112255 |
| Date | 30 April 2021 |
| Creators | Murray, Jacob Hadley |
| Contributors | Chemistry, Santos, Webster L., Carlier, Paul R., Etzkorn, Felicia A., Tanko, James M. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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