br STAR Methods br Acknowledgments We

STAR★Methods
Acknowledgments We would like to thank Dr. Xiping Cheng for help with the immunohistochemistry staining, Qi Su for help with gene expression analysis, and Dr. Chaoying Liang at the UTSW Genomics Microarray Core facility for assistance on RNA-seq. W.-H.L. was supported by grant awards from NIH (R01 GM07593), JDRF (17-2013-494), and Welch Foundation (I-1902). J.K., H.O., K.C., Y. Xin, E.N., R.H., J.L., B.Z., A.J.M., G.D.Y., and J.G. are employees and shareholders of Regeneron Pharmaceuticals.
Type 2 Diabetes Mellitus (T2DM) is a growing worldwide epidemic currently affecting an estimated 300million people. Despite a number of diabetic therapies available nowadays, there remains a significant unmet medical need for additional therapies. T2DM is partially characterized by elevated and dysregulated hepatic glucose production (HGP). Glucagon is a 29-amino (E)-2-Decenoic acid peptide that acts as a major counter-regulatory hormone to insulin, and stimulates gluconeogenesis and glycogenolysis in liver and thus increasing HGP. Studies in type 2 diabetic subjects have demonstrated a causal role for glucagon in promoting excessive glucose production. An inappropriately high rate of HGP is the predominant cause of fasting hyperglycemia and a major contributor to the postprandial hyperglycemia characteristic of T2DM. It is therefore postulated that blocking the action of the glucagon receptor would lead to improved glycemic control in T2DM patients. With this, there have been many Letters detailing efforts to identify both small and large molecule glucagon receptor antagonists (GRAs) as potential treatments for T2DM over the last two decades., Clinical data on one of the early GRAs, Bay 27-9955() was reported and an acute effect was observed with the compound in blocking glucagon-induced glucose increase in healthy volunteers. A series of β-alanine acid containing urea based GRAs such as NNC 25-0926 () was later reported by Novo-Nordisk., , Various scaffold changes replacing the urea core while keeping the β-alanine moiety by many groups have since been reported in patent applications and literature (e.g., GRA reported by Pfizer). Prominent examples of this include several GRA clinical candidates, such as MK-0893 (, Merck) and likely LY2409021 (Eli Lilly). With the continued interest in the area of developing novel GRAs as a potential treatment for T2DM, we designed a novel series of GRAs possessing an indazole or an indole/azaindole core (, ) as intrigued by the presence of a unique 5-naphthylpyrozole moiety in the core structure of MK-0893. In this Letter, we would like to discuss the synthesis and the structure–activity relationship (SAR) studies of this series of GRAs, leading to the identification of several potent compounds which demonstrated excellent in vitro and in vivo profiles. The synthesis of the indazole-based GRAs is shown in .Treatment of 5-bromo-, 4-bromo-, or 3-bromo-2-fluorobenzaldehydes with methoxyamine in the presence of KCO, followed by refluxing with hydrazine in DME afforded corresponding 5-, 6-, or 7-bromoindazoles in good yields. Iodination of with iodine/KOH in DMF provided 3-Iodoindazoles in excellent yields. Bromination at the benzylic position of 4-alkylbenzoic acids was achieved with NBS and benzoyl peroxide, and the resulting was coupled with β-alanine ethyl ester to afford amides . Indazoles were then alkylated at the -1 position with benzylbromides in the presence of CsCO in DMF to provide . Suzuki couplings of with ArB(OH) catalyzed by Pd(PhP)Cl preferentially at the 3-postion of the indazole (resulted in the partial hydrolysis of ethyl ester), followed by a second Suzuki coupling with ArB(OH) at the 5-, 6-, or 7-position of the indazole core under similar conditions were then performed. The sequential Suzuki couplings provided desired bis-substituted indazoles – in moderate to good yields over two steps. To facilitate the chiral resolution of the racemic compounds, the acids were converted to methyl esters by simple treatment with TMSCHN in MeOH/THF, and re-hydrolyzed back to acids after chiral separation.