Our synthetic approach to GPR antagonists was designed

Our synthetic approach to GPR55 antagonists was designed so that many different structures could be accessed to rapidly explore initial SAR, along with validating or modifying our current model (). The synthesis begins with the coupling of a carboxylic rac protein to 4-piperidone by first forming the acid chloride (). The different acids chosen, based on the initial hit, modify the electronics and sterics of this section of the molecule. Relative to ML191, compound reduces the steric impact, increases the electron-density in the aromatic ring, and compounds and decrease the electron-density. Compounds and were selected to examine the influence of steric bulk at the position of the cyclopropane ring. The largest change in overall structure relates to the 1-naphthoic acid derivative (). Although the naphthalene ring is structurally different, this analogue can position the distal aromatic ring in a similar position as the phenyl rings of the other analogues since the bond angle for the Cα will be similar to that of the cyclopropane analogues, however, this structure is much flatter. With a handful of acylated piperidones prepared, the final two steps first involved a reductive coupling of aryl hydrazides (–) with the previously synthesized piperidones (–) to yield hydrazides (). These compounds were then cyclocarbonylated using triphosgene to yield oxadiazolones . The reductive coupling reactions proceeded smoothly but the products of that step were often unstable to silica gel chromatography. Therefore, the unpurified products were treated with triphosgene without further purification. This modification of the synthesis typically improved the yields of the final compounds (see for individual yields). Similar to the cyclopropane starting materials (–), the hydrazides (–) were selected to probe the electronic and steric opportunities in the binding site. Based on the current model (), the aromatic ring adjacent to the oxadiazolone is involved in an interaction with M3.36(105) and F6.48(239). Additionally, the oxadiazolone contributes as the key interaction between the basic carbonyl oxygen with the ammonium of K2.60(80). Thus, electron rich aromatic rings adjacent to the oxadiazolone should make the carbonyl oxygen more basic and strengthen this interaction. A targeted exploration of the SAR of all six acids (–) with hydrazide and all seven hydrazides (–) with acid ( and ) was performed instead of synthesizing and exploring the biological activity of all 42 permutations of the six acids and seven hydrazides. Acid and hydrazide were chosen as the constants since these were the most simplified pieces consisting of an unsubstituted phenyl ring. Unfortunately, there were solubility issues with some of the compounds (e.g., and ), so additional combinations were required to elucidate the effect of the different areas of the scaffold. Compounds were initially screened via an image-based cell assay to identify antagonist activity. The rationale for using the β-arrestin recruitment assay was to provide a fair comparison of IC values since our initial report employed this assay., Briefly, U2OS cells overexpressing GPR55 and βarr2-GFP were exposed to LPI (6μM; EC) resulting in the recruitment of β-arrestin. Antagonist activity was evaluated by ligand-mediated inhibition of LPI-induced receptor activation. This strategy quickly identified the compounds that had IC values higher than 15μM which were excluded from further analysis (). Concentration response curves were generated for compounds that were active at concentrations below 15μM employing both the image-based β-arrestin recruitment assay and the DiscoveRx PathHunter® chemiluminescent β-arrestin complementation assay. In the DiscoveRx PathHunter® system, CHO-K1 cells stably expressing GPR55 (fused with a β-galactosidase enzyme fragment), and β-arrestin (fused to an N-terminal deletion mutant of β-galactosidase) were used to quantitate the inhibition of LPI-induced β-arrestin activity. (, ). Hence, antagonist activity was evaluated through the use of two differential means of β-arrestin quantitation, in two different cellular backgrounds (see , Biological Assay). IC values were similar in both methodologies.