All analogs were tested for inhibition of both AC1-and AC8-mediated Ca 2+ /CaMstimulated cAMP production in a cellular assay. For these activity assays, HEK293 cells had endogenous AC3 and AC6 isoforms knocked out using CRISPR-Cas9 followed by subsequent stable transfection and overexpression of AC1 or AC8 isoforms. SAR was informed based on potency against AC1-mediated cAMP production. However, in terms of evaluating selectivity against AC8 a simple comparison of IC50 values does not adequately convey this measure. This is because in the concentration-response curves it was observed that inhibition often reaches baseline levels for AC1 at the higher molecule concentrations while AC8 is never fully inhibited by any analog (representative curves for 1 shown in Figure ). This indicates that the scaffold displays a difference in maximal inhibitory efficacy between isoforms; therefore, merely evaluating the AC8 IC50 to assess selectivity is misleading as the IC50 is generated from the relative maximum and minimum cAMP signal for AC8. Case in point, analog 1 has a calculated IC50 value of 1.6 µM against AC8 even though it clearly is never fully inhibited. Thus, a more accurate measure of selectivity for AC1 over AC8 would be the evaluation of the percent inhibition of AC8 at the IC90 of AC1 activity (Figure , black arrow). Therefore, the AC8 activity will be referred to in this context. All AC1 IC90s are reported in Table and concentration response curves for all molecules against AC1 and AC8 are included in the supporting information. Initial hit compound 1 was identified in the primary screen as a potent, selective inhibitor of Ca +2 /CaM-stimulated AC1 activity. After re-synthesis and validation, it was found to have a cellular IC50 of 1.4 µM versus AC1-mediated cAMP production and 46% inhibition versus AC8mediated cAMP production at the AC1 IC90. This AC1 potency was comparable to, or an improvement upon, the prior art for AC1 inhibitors and this scaffold was prioritized for optimization. Removal of the fluorine to yield unsubstituted phenyl derivative 6 displayed slightly reduced potency with an IC50 of 2.4 µM versus AC1. We then evaluated a group of analogs containing phenyl ring replacements in compounds 7-9. The phenyl ring was removed leaving an acetamide in compound 7, which showed a complete loss of activity against both ACs. Two bioisostere substitutions, a furan (8) and a pyridine (9), were assessed next. The furanyl substitution 8 was tolerated, albeit with reduced potency (AC1 IC50 value of 6.7 µM), whereas the pyridinyl analog 9 displayed similar activity to the phenyl (AC1 IC50 value of 1.2 µM) indicating the pyridine may be a suitable replacement for the phenyl without detriment to AC1 activity.