Structure-Activity Relationship. We initially targeted oxyberberine natural products 2 and 3 with higher oxygenation patterns because they have not been studied in a biological context and 2 had not been synthesized previously. In vitro kinase activity assays using the AMPK (α1/β1/γ1) kinase enzyme system (presented as IC50 data) and in vivo assays using the human intestinal epithelial cell line HT29 2c revealed that altering the cationic isoquinolinium moiety characteristic of berberine (1) for the neutral isoquinolone stimies AMPK activation activity (Schemes 1b and 1c). Highly oxygenated alkaloids 2, 3, and 14 were poorly active or inactive in vitro. Removal of the two methoxy groups from the D ring resulted in a compound that inhibited AMPK with an IC50 of 85.3 μM. Fully reducing the enaminone motif of the isoquinolone resulted in racemic tertiary amine 17 with similar inhibitory potential. Rendering the D-ring electrondeficient with a cyano group (e.g., 18) gave an inactive compound. Stepharotudine-type compounds 4, 19, and 20 with guaiacolic A-rings exerted the greatest activities with unnatural 20 displaying an IC50 of 2.5 μM. This data suggests that the protoberberine derivatives examined in this study exhibit a distinct mechanism of action compared to berberine (1) and can directly inhibit AMPK. Western blot analysis additionally supports the inhibitory activities of the protoberberine analogs against phosphorylated AMPK (Scheme 1d). While 8-oxypseudopalmatine (3) was inactive in vitro, it produced the greatest inhibition when subjected to whole cells by Western blot. Presumably, demethylation of one or more of the methoxy substituents in cells generated a guaicolic derivative that exerts greater potency. We were able to test the B-ring distortion analogs (4-iso and 20-iso) by Western blot analysis and found them to also inhibit p-AMPK (see SI). Thus, deviating from planarity does not substantially impact AMPK modulation. Collectively, the data suggest that the A-ring guaiacolic -OH group and an electron-rich D-ring are important in enhancing AMPK inhibition. Our study is the first to uncover the AMPK inhibition potential of protoberberines that have long been studied for their activation properties, while elucidating structural features that govern bioactivity.