k j and d were then submitted to

15k, 15j and 14d, were then submitted to metabolic stability studies in human and mouse liver microsomes and permeability test with Caco-2 cells (Table 7). 15k, 15j and 14d all retained excellent permeability (Papp value greater than 15.2 × 10−6 cm/s) and displayed modestly improved metabolic stability, compared with 2. 14d with 4-fluorine phenylpiperazine as tail demonstrated the best stability in mouse liver microsomes. When the tail and linker were replaced with (S/R)-1,2,3,4-tetrahydro-1-naphthylamine and a benzene ring (15k) there was a markedly improved stability in human liver microsomes. The S,R-isomeres (15k) was more stable than the S,S-isomeres (15j) in both human and mouse liver microsomes, indicating that the stereochemistry of the tail had little effect on potency but great impact on metabolic stability. The PK and the tissue distribution profiles of 15k, 15j, 14d and 2 (Table 8) in male ICR mice were assessed. 15k, 15j, 14d all exhibited prolonged half-lives and improved plasma exposure. 15k achieved the highest concentration (15.45 μg/mL) in plasma among four compounds. The observation that 15j (S,S-isomers) and 15k (S,R-isomers) differed substantially in peak times and peak plasma concentrations indicated that the stereochemistry of the tail significantly affected absorption. Compared with 2, 15k had 5.4-fold, 4.1-fold and 15.5-fold improvement in plasma exposure, half-life time and AUC (area under the curve), respectively. Tissue distribution assays showed that 14d, 15j and 15k had much lower CNS exposure (B/P = 0.018, 0.006 and 0.011 respectively) than 2. We also examined the distribution of compounds to the liver, based on the report that TAK-875 had 3 times higher distribution to the liver than to plasma [35]. 15j and 15k displayed lower L/P compared with 2. However, 14d was not studied any further because of safety concerns associated with its 15-times higher distribution to the liver than to plasma. 15k, with promising PK and safety profiles, was progressed to in vivo efficacy studies, where it demonstrated a significant decrease (P < 0.001) in blood Calpain Inhibitor XII level following a 2.5 g/kg oral glucose load in the oral glucose tolerated test (OGTT) (Fig. 4).
Conclusion We developed a series of amide derivatives driven by the moderate CNS exposure, high in vitro clearance and limited oral exposure of 2. By inserting a carbonyl at the position prone to N-dealkylation metabolism and opening up the indene, as well as introducing a nitrogen atom to reduce LogP and elevate tPSA, we obtained 13c with improved physicochemical properties and liver microsomal stability. Further SAR research and optimization yielded 15k, which exhibited the best in vitro activity, excellent permeability and markedly improved in vitro stability. Furthermore, studies demonstrated that 15k possessed superior PK properties in vivo, lower distribution to brain and liver than to plasma compared with LY2881835, supporting that 15k is more likely to avoid the undesired effects in the CNS and liver. In conclusion, our results disclosed an orally efficacious compound 15k, which represents a promising lead compound for developing a safe antidiabetic drug that acts via activation of GPR40.
Experimental section
Abbreviations AUC, area under curve; BBB, brain-blood-barrier; (BOC)2O, di-tert-butyl-dicarbonate; B/P, total brain-to-plasma drug distribution ratio; CCl4, carbon tetrachloride; CL, clearance; CNS, central nervous system; Cmax, maximum plasma concentration; DCM, dichloromethane; DIPEA, N-diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformadide; DMSO, dimethylsulfoxide; FFAR1, free fatty acid receptor 1; FFAs, free fatty acids; FLIPR, Fluorometric Imaging Plate Reader; GLP-1, glucagon like peptide 1; GPR40, G-protein coupled receptor 40; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HPLC, High Performance Liquid Chromatography; ICR mice, Institute of Cancer Research mice; L/P, liver-to-plasma drug distribution ratio; NADPH, Nicotinamide Adenine Dinucleotide Phosphate; NBS, Nbromosuccinimide; PK, pharmacokinetic; SAR, structure-activity relationship; T, half-life time; T2DM, type 2 diabetes mellitus; tPSA, total polar surface area.