While previously reported optimizations on compound focused

While previously reported optimizations on ap-1 focused on the phenyl ring A () and substituents on isoxazole ring, other alternatives to replace isoxazole ring were never explored. Herein, we propose to replace the isoxazole with its bioisosteric substituted phenyl ring B () to explore new chemical space and improve EPAC inhibitory potency. Docking studies of compound into the cAMP binding domain of active EPAC2 suggested that 3-chloro phenyl ring A and -butylisoxazole occupy two hydrophobic pockets and play important roles for their interaction. Compound was synthesized and gave an IC value of 13.3 µM, showing the phenyl bioisosteric replacement was a viable route forward. The first series of compounds were designed to keep the -butyl phenyl group B intact and the synthetic route is depicted in . Starting material methyl 4-(-butyl)benzoate () was reacted with CHCN in the presence of CHLi to give 3-(4-(-butyl)phenyl)-3-oxopropanenitrile (). Anilines were transformed into corresponding diazo salts and coupled with in the presence of NaOAc to produce compounds and ∼ in good yields. The second series of compounds were designed to replace -butyl group on phenyl ring B, and the synthetic protocol is depicted in . 3,5-Dichloroaniline was transformed into its corresponding diazo salt and coupled with different 3-oxo-3-phenylpropanenitriles in the presence of NaOAc to give compounds – in moderate to good yields. All the structures and purity of synthesized compounds were confirmed by H NMR, C NMR and HR-MS. Compounds were then evaluated for their abilities to inhibit EPAC1 and EPAC2-mediated Rap1b-bGDP exchange activity using purified recombinant full-length EPAC1 and EPAC2 proteins. For promising compounds, the IC values against both EPAC1 and EPAC2 were determined ( and ). Compound was used as the reference compound for comparison with IC values of 10.8 µM and 4.4 µM against EPAC1 and EPAC2, respectively. Based on our previous SAR study, 3-Cl or 3-CF on phenyl ring A which resides in a hydrophobic pocket P1 is favorable for EPAC binding. Thus, for most newly designed compounds, we chose to retain these substituents. Compared to compound , compound with an additional 5-Cl substituent has 2.5-fold improvement (IC = 5.4 μM) on EPAC1 inhibitory activity and 11-fold improvement (IC = 2.5 μM) on EPAC2 inhibitory activity. In addition, substituent at 5-position is more favorable than that with a same group at 4-positon on this phenyl ring A (compound vs , and compound vs ). Triple substituents at 3,4,5-position displayed slightly decreased EPAC inhibition (compounds and ), but compound has a 4.6-fold EPAC2/EPAC1 selectivity. 3,5-Di CF substituted compound showed similar EPAC inhibitory activities to those of compound . The first round of optimization illustrated that all compounds obtained via bioisosteric replacement of -butyl isoxazole ring with -butyl phenyl group retained both EPAC1 and EPAC2 inhibitory activities, while nearly half of them displayed more potent inhibitory activities than reference compound . 3,5-Di Cl substituents on phenyl ring A are favored and compound exhibited the most potent EPAC1 and EPAC2 inhibitory activities. Thus, we kept this fragment intact and further modified substituents on phenyl ring B which occupies the other hydrophobic pocket P2 (). We investigated 3-Cl () and 4-Cl () first, but both are detrimental to EPAC inhibition. It appears that the bulk size of substituents on phenyl ring B is critical. We then explored electron donating group 4-OCF () and electron withdrawing group 4-COMe (). Interestingly, neither of them showed increased inhibition against EPAC1 compared to compound . Introduction of 4-Ph and 3,4-di OMe substituents on phenyl ring B led to compounds and which have a complete loss of EPAC inhibitory activities. The various rings including piperidine (), morpholine () and cyclohexane () were also investigated, but none of them displayed more potent EPAC inhibition than compound . However, compounds ∼ with fused rings (e.g. 1,2,3,4-tetrahydronaphthalene, naphthalene and quinoline) all displayed improved EPAC inhibitory activities when compared to afore-mentioned compounds that have different substituents on phenyl ring B. Among them, compound exhibited the most potent EPAC inhibitory activities with IC values of 3.6 μM and 1.2 μM against EPAC1 and EPAC2, respectively. In , examples of dose-response curves are shown for compounds , , and .