Drug combinations are important measures to improve antitumor efficacy and overcome tumor resistance in clinic.Compared with drug combinations,multi-target drugs can effectively avoid adverse drug-drug interactions and improve patients’ compliance while exerting similar activities,which has become a hot topic in medicinal chemistry.Tumor invasion and metastasis are one of the reasons why malignant tumors are difficult to be cured.Many studies have revealed that aminopeptidase N(APN)can degrade extracellular matrix(ECM)and then promote tumor metastasis.Moreover,an increasing number of studies have indicated that overexpression of APN is involved in various cancers.Therefore,APN is regarded as an important antitumor target.The PI3K/AKT signaling pathway is one of the important signaling pathways which plays a significant role in regulating cell proliferation,cell cycle,and apoptosis.Activation of this pathway is common in many human cancers.Protein kinase B(AKT,PKB)plays a crucial role in PI3K/AKT signaling pathway.Thus,AKT is also an important antitumor target.In recent years,many studies have shown that APN inhibitors and AKT inhibitors have synergistic effects in some solid tumor cells.For example,combination of bestatin and AKT inhibitors showed better inhibitory activity in melanoma cells.Bestatin can also inhibits the activation of the CD13/EMP3/PI3K/AKT/NF-κB pathway to overcome cisplatin(CDDP)resistance in gastric cancer cells.Therefore,APN/AKT dual-target inhibitors are expected to exert more potent anti-cancer activity than single-target drugs.Based on the pharmacophores of AKT inhibitor AZD5363 and APN inhibitor bestatin,we used the principle of molecular hybridization to design and synthesize two series of APN/AKT dual-target inhibitors containing nineteen novel compounds,which were progressed to in vitro evaluation.The results of in vitro APN inhibitory assay revealed that most of the target compounds showed good inhibitory activities against APN.Specifically,compounds(S)-5a,(S)-5b,(S)-5c,(S)-5d,(S)-5e,(R)-5f,(R)-5g,(R)-5h,(R)-5j,10a,10b,10c,10d,10e,10f,12a and 12b exhibited more potent APN inhibitory activities than the positive control bestatin.Among them,the most potent compound 12b(IC50=0.05 μM)was over 70 times more potent than bestatin(IC50=3.64 μM).Structure-activity relationship study revealed that the R configuration of the R substituent is benefit to APN inhibition.Therefore,the R substituent was fixed as R configuration in the following structural modification and optimaization.As for the in vitro AKT1 inhibitory assay,some target compounds displayed promising AKT1 inhibitory activities with compounds(R)-5f and(R)-5h possessing IC50 values of 0.12 μM and 0.27 μM,respectively.It is worth noting that compound(R)-5h showed balanced inhibition against APN and AKT1(IC50 values were 0.27μM and 0.21 μM,respectively).HUVECs tubular structure formation model confirmed the excellent APN inhibitory activities of compounds(R)-5f and(R)-5h at the cellular level,which were much better than that of bestatin.Western Blot analysis demonstrated that compound(R)-5h can effectively inhibit the phosphorylation of GSK3β(the substrate of AKT)in a dose-dependent and time-dependent manner.Subsequently,we selected MDA-MB-468 and AGS cell lines for in vitro antiproliferation assays.However,most of the target compounds did not show significantly antiproliferative activity.Finally,molecular docking was used to simulate the binding modes of compound(R)-5h with APN and AKT1,respectively.The results showed that(R)-5h can occupy both the active site of APN and the ATP binding pocket of AKT1,which not only supported our design strategy,but also offered basis for further structural optimization. |