br Materials and methods br

Materials and methods
Results
Discussion
Acknowledgments This work was financially supported by the University of Tehran, Iran. The authors thank all the teams who worked on the experiments and who provided technical assistance in the laboratory during this study. We also thank the anonymous reviewers whose critical comments helped in improving the manuscript.
Obesity affects life quality and endangers the general health of a large population, and it may replace tobacco as the number one health risk for developed societies. It is estimated that about 127 million adults in the United States are overweight or obese, and approximately 300,000 deaths per year are directly attributable to obesity, mainly due to associated heart disease, diabetes, or cancer. Several other medical conditions can also be attributed to obesity, including asthma, sleep apnea, arthritis, reproductive complications, and psychological disturbances. Over the years, many therapeutic interventions have been used for the treatment of obesity, but most of them are either complicated with safety issues or limited in patient population. Ghrelin, a 28 amino Imeglimin peptide hormone bearing an octanoyl side chain at the third amino acid from its N-terminus (serine 3), is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). It is synthesized primarily in the stomach and found in the circulation of healthy humans. The GHSR was identified to be a G protein-coupled receptor (GPCR) located predominantly in the pituitary gland and the hypothalamus. Ghrelin levels in plasma are influenced by nutritional status and regulate growth hormone (GH) secretion, appetite and fat deposition. Administration of ghrelin to rats results in weight gain as a consequence of changes in energy intake and fuel utilization. Moreover, systemic ghrelin administration in humans stimulates sensations of hunger and induces overeating. Based on these findings, ghrelin is believed to play a crucial role in the regulation of appetite and body weight, and GHSR antagonists are expected to reduce appetite and food intake, and may provide treatment of eating disorders and obesity. Thus, we and others have been interested in identifying GHSR antagonists for the treatment of obesity. Herein, we report the discovery and optimization of piperazine-bisamide based GHSR antagonists. High throughput screening of our chemical library was conducted with an aequorin flash luminescence (Aeq) assay using CHO cells stably expressing human GHSR. This assay measured the inhibition of compounds on the intracellular calcium increases induced by ghrelin (1.0μM) and identified the piperazine-bisamide () as a potent antagonist (IC=100nM) for the GHSR receptor. However, further study of in a human inositol phosphate (IP) accumulation assay demonstrated an 18% increase in IP at 10μM relative to ghrelin’s maximal response., Therefore, efforts to optimize the potency of compounds in the aequrin assay while reducing their partial agonist properties observed with the IP assay were pursued. All compounds tested in the human or rat IP assays were evaluated at 0.1μM, 1.0μM, and 10μM. However, for purposes of comparing their maximal agonistic response in IP assays we chose to report the compounds activity at 10μM. The synthetic route to obtain these piperazine-bisamide compounds is outlined in . Following route , treating commercially available (-butyl piperazine-1-carboxylate:=1, or -butyl 1,4-diazepane-1-carboxylate:=2) with 4-biphenyl carboxylic acid or 3′-methoxy-biphenyl-4-carboxylic acid in the presence of HBTU and Hünig’s base in DMF followed by boc-group deprotection afforded intermediate , which upon second amide coupling with the corresponding aryl-carboxylic acid provided –, , and , respectively. Reductive amination of the amine intermediate with indole-6-carboxylaldehyde afforded in 54% yield. Starting from , this synthetic approach provided access to bromo-substituted intermediate , which after either Suzuki or Stille coupling reactions gave compounds –, ,,,, , , –, or ,. The intermediate involved in this route also provided convenient access to compound after amide coupling with the corresponding commercial carboxylic acids. In contrast to this approach, while route also shared the same starting material (either the or enantiomer), it switched the order of the amide bond formation and thus provided compounds –. We also designed a synthesis for the middle B-ring replacements with heterocycles as shown on route . Pre-installation of the heterocyclic B-ring onto the biaryl tail of the molecule through Suzuki coupling of the Br- or Cl-heteroaryl methyl carboxylates with the corresponding boronic acids followed by saponification provided intermediate carboxylic acid . Coupling of with offered the corresponding compounds –, , and in moderate to good yields.