Majed Alharbi

AMP‐activated Protein Kinase (AMPK) has conflicting roles in solid tumor progression as it shows anticarcinogenic activities in healthy cells while it may help the existing cancer cells to survive through certain stress conditions such as metastasis, hypoxia, and dormancy. These cells are usually present in the core of the solid tumors. To explore AMPK inhibitors and their anticancer activities, three compounds related to N ‐benzyl‐[4‐(phenylamino)aryl]acetamide scaffold demonstrated high potency as α1/β1/γ2 AMPK allosteric inhibitors (IC 50 range 7.77–13.75 nM). As an example, the compound N ‐(6‐(2‐(benzylamino)‐2‐oxoethyl)pyridin‐3‐yl)‐3‐methoxybenzamide (MAK‐11) inhibited cancer growth at IC 50 values 0.148 μM (MCF7, monolayer), 0.097 μM (T47D, monolayer), 19.58 μM (MCF7, spheroids) and 3.72 μM (T47D, spheroids). It was also found that MAK‐11 caused increases in spheroid model dead cells (36.5 % and 32.1 %), induced mitochondrial depolarization (34.0 % and 36.8 %), and elevated ROS levels (24.6 %% and 32.1 %) for MCF7 and T47D, respectively. We also profiled the drug‐likeness of compounds and confirmed that they do not inhibit HSF (non‐cancerous cell lines). It was noticed that monolayer (2D) assay results are not parallel to the spheroid (3D) results as MAK‐11 was 97 times lower than MAK‐6 in T47D 2D assay, while it was twice as potent as the same compound in the 3D assay. Therefore, we propose that AMPK inhibitors should be tested on 3D tumor models that are similar to the environment with the core of tumor cells.