The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R₇ and R₈ A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC₅₀ values as low as 210 nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R₇ cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.

Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits IκB kinase ε (IKKε) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes the inhibitory effects. IKKε is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKKε and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.