Based upon these findings on portions A and
Based upon these findings on portions A and B, novel scaffolds of EP4 antagonist, and (R & R=()-Me; =Cl; R=H), shown in , were identified. We next focused on optimizing portion C of these scaffolds. We utilized for an alternative synthesis of nicotinamide scaffold , which is quite effective for derivatization of portion C. Thus nicotinic Nocodazole was coupled with -butyl 4-[(1)-1-aminoethyl]benzoate using EDCI/HOBt condition to give 2-chloro-nicotinamide . Nucleophilic displacement of 2-chloro-nicotinamide with appropriately substituted phenols afforded 2-phenoxy-nicotinamides . A final acid hydrolysis of -butyl ester moiety in intermediates under acidic conditions furnished respective benzoic acids . A SAR of the representative analogs of portion C are shown in . Substitution at the -position of the benzene ring in portion C of nicotinamide scaffold () with a fluorine or chlorine atom enhanced the functional activity as compared with the corresponding - or -positions ( vs or , vs or ). Therefore, the -substitution was explored in more details. The introduction of small and electron-withdrawing substituents such as fluoro, chloro, cyano, and trifluoromethyl at the -position of the benzene ring was found favorable (, , , and ). Among the four analogs, -fluoro analog was the most active compound. Although di-fluoro substituted analogs such as 3,4-di-fluoro-, 2,3-di-fluoro-, 2,5-di-fluoro-, and 3,5-di-fluoro-analog exhibited IC values of less than 50 nM in the functional assay (data not shown), these analogs did not surpass the functional activity of . On the other hand, the substitution with electron-donating groups such as methoxy or methylthio at the -position appeared to be less favorable ( and ). Trifluoromethoxy group at the -position was not tolerated at all (). Replacement of the phenyl group in portion C by 3-pyridyl group decreased the functional activity ( and ). Incorporation of hydrophilic functionality such as carboxamides, sulfones, and sulfonamides on the benzene ring also caused substantial decrease in intrinsic activity (data not shown). A similar tendency in the SAR studies of the analogs with benzamide scaffold () were observed, however approximately 2-fold less potent than the corresponding nicotinamide scaffold analogs ( vs , vs ) with the exception of , which displayed equivalent activity to . Key compounds (, , and ) that exhibited potent EP4 functional activity were evaluated for their selectivity against the other EP receptor subtypes. These potential compounds showed sufficient EP4 selectivity. Furthermore, compounds , , and had high permeability in the Caco-2 assay. Based on these SAR studies, six compounds which exhibit IC values <40nM in the human EP4 functional assay were selected for further characterization. shows comparative data of the six compounds. All the compounds showed high metabolic stability in HLM (>120min). Although the intrinsic activity of 3-cyano analog was sufficient to progress to the next stage, was found to be positive in the micronucleus test and suffered from poor membrane permeability and high efflux ratio in the Caco-2 assay at equivalent pH condition ( A to B: 0.644×10cm/s, B to A: 25.8×10cm/s, Efflux Ratio: 40). It was found that the 3-chloro analog was not a pure antagonist but a partial agonist, therefore it is assumed that the functional antagonism of (IC: 21.8nM) was caused by its partial agonism. The remaining four compounds (, , , and ) were subsequently re-evaluated for their antagonistic potencies (p values) against human EP4 receptor and oral activity in the rat carrageenan induced mechanical hyperalgesia model. Among them, showed the highest potency which met the backup candidate criteria (p: 8.97), and was selected for preclinical development with code AAT-008. As shown in , AAT-008 () selectively antagonized the prostaglandin receptor EP4. AAT-008 inhibited [H]-PGE binding to human EP4 receptor with a value of 0.97nM and had more than 1000-fold selectivity over other prostaglandin receptors, such as EP1, EP2, EP3, DP, FP, IP, and TP. AAT-008 showed potent binding affinity for human, rat, and dog EP4 receptors, with values of 0.97, 6.1, and 38nM, respectively. In the human EP4 receptor highly expressed in HEK293 cells, AAT-008 significantly suppressed PGE-induced elevation of intracellular cAMP level in a PGE-competitive fashion with a p value of 8.97 (1.1nM). Compared with grapiprant (), AAT-008 was approximately 4-fold more potent in the functional assay.