We next determined the selectivity

We next determined the selectivity profile of the most potent GPR40 agonists (4k–n) against other free fatty Cathepsin K Activity Fluorometric Assay Kit receptors (FFA3/GPR41, FFA2/GPR43 and FFA4/GPR120). FFA2 and FFA3 agonists have a preference in binding short-chain fatty acids while FFA1 and FFA4 have a higher affinity to medium- and long-chain fatty acids.19, 20 As it is seen from the activity data against this panel of GPCRs, the four lead compounds displayed high selectivity for FFA1 (Table 2). In order to better understand the dramatic influence of the benzyl substituent in the 1-oxa-9-azaspiro[5.5]undecane portion of the compounds (compared to the unsubstituted derivative), we have docked 4l and 4s in the FFA1 binding site (Fig. 3). Both compounds occupy similar positions within the binding cavity, forming the critical hydrogen bonds with R1835.39, R2587.35, Y913.37 and Y2406.51. The propanoic acid moiety may be further stabilized by a network of π–π interactions that could occur between F883.34, F1424.61, W174EL2 and F873.33 of the receptor and the aromatic rings of each compound. However, there are significant differences in the positioning of the spiro-pyran moiety, which is likely responsible for the observed differences in the potency of 4l (EC50=0.055μM) and 4s (EC50>20.00μM). In 4s, this moiety positions toward the extracellular side while, in contrast, the potent 4l adopts a position, where 1-oxa-9-azaspiro[5.5]undecane faces toward the intercellular side (A). Here, the terminal benzyl substituent positions within a favorable hydrophobic cavity forming numerous hydrophobic interactions with L542.51, L1354.54 and V813.27, while also having π–π stacking interactions with W1314.50 (B). Together these contacts drive the benzyl-substituted 1-oxa-9-azaspiro[5.5]undecane moiety deep into the phospholipid bilayer, which might be beneficial for agonist activity. To appreciate the positioning of the lipid bilayer and its possible interactions with the ligand tails, we used the Orientation of Proteins in Membrane (OPM) database and PPM webserver (REF: 27) to align and predict the position of FFA1 in a membrane (A). The position of 4s within the binding cavity is seemingly tolerable, however, the ligand stability is likely compromised by the electrostatic interactions between the positively charged ammonium of its spiro-pyran motif and the negatively charged phospholipid head. Here, the fluidity of the lipid bi-layer coupled with these strong electrostatic interactions may destabilise the binding conformation, which likely attributes to the low activity of the compound. In contrast, the benzyl substituent of compound 4l favors the more hydrophobic environment (B), positioning at the interface between the lipid tails and protein which negates the detrimental attraction between the phosphate heads and positively charged ammonium. Since we were able to reach well into submicromolar potency range with our pilot set of 19 compounds (4a–s), we were also keen on assessing the preliminary ADME profile of the most active compounds from this set (4k–n). On examination of the data presented in Table 3, several general observations can be made. All four compounds displayed satisfactory aqueous solubility and Caco-2 permeability, which confirmed the new chemotype was promising from further development prospective. The relatively high plasma protein binding observed for 4k–n perhaps is not surprising for these highly lipophilic compounds and may not present a significant development obstacle provided the rate of dissociation from plasma proteins is sufficient to maintain the required plasma levels (vide infra). The principal weakness of the series studied herein was its low metabolic stability determined by incubation of compounds 4k–n in the presence of mouse liver microsomes. Indeed the half-life of 4l–m was around 10min with unacceptably high clearance. This could be attributed to the presence of two heteroatom-substituted benzylic positions or β-unsubstituted propanoic acid moiety, which could be prone to metabolic oxidation.