3CAI is a potent and specific AKT1 and AKT2 inhibitor which showed significant inhibition of AKT in an in vitro kinase assay and suppressed expression of AKT direct downstream targets such as mTOR and GSK3β as well as induced growth inhibition and apoptosis in colon cancer cells.

3CAI, a potential AKT inhibitor, exhibits significantly higher potency in inhibiting AKT1 compared to PI3K (60% inhibition at 1 µM vs 10 µM, respectively). In experiments with HCT116 and HT29 colon cancer cells treated with 3CAI (4 µM), I3C, or an AKT inhibitor in 1% FBS/McCoy's 5A (for HCT116) over four days, 3CAI notably increased apoptotic cell numbers versus untreated controls. Additionally, 3CAI markedly reduced AKT-mediated phosphorylation of mTOR (Ser2448) and GSK3β (Ser9) in a time-dependent manner, while also elevating pro-apoptotic proteins p53 and p21 after 12 or 24 hours of treatment. In vitro kinase assays revealed that 3CAI solely inhibits AKT1 kinase activity without affecting the activities of MEK1, JNK1, ERK1, and TOPK at 1 µM. Its capability to suppress AKT1 and AKT2 activities indicates a dose-dependent effect, with a profound impact on downstream AKT targets and the induction of apoptosis.

To assess the in vivo antitumor efficacy of 3CAI, athymic nude mice with HCT116 cancer cell-induced tumors on their right flank were orally administered 3CAI at doses of 20 or 30 mg/kg, I3C at 100 mg/kg, or a vehicle control, five times weekly for a duration of 21 days. The higher dosage of 3CAI (30 mg/kg) was found to notably suppress the expression of AKT-target proteins in tumor tissues. Furthermore, this dosage significantly reduced tumor growth by 50% compared to the control group (p<0.05), without inducing noticeable side effects or substantial weight loss in the mice, suggesting a good tolerance to the treatment.