| Aminopeptidase N (APN/CDA3) is a membrane binding exopeptidase belonging to type Ⅱ zinc-dependent metalloprotease, which is widely expressed as an integrin dimer on the surface of various mammalian cell. As an exopeptidase, APN functions ubiquitously in different peptide metabolism pathways, so it plays pivotal roles in many physiological processes. Interestingly, APN is observed to be over-expressed in many tumor cells, and plays a key role in tumor angiogenesis, invasion and metastasis process. Accordingly, APN is considered as an important target for anti-tumor research.To date, many natural or synthetic APN inhibitors have been reported. Natural APN inhibitors include Bestatin, Probestin, Phebestin, AHPA-Val, Lapstatin, Amastatin, Curcumin, and so on. Synthetic APN inhibitors include α-aminoaldehydes, a-aminophosphiric acids, α-aminoboronic acids,β-amino-thiols, L-isoglutamines, L-lysine derivatives, L-arginine derivatives, cyclic-imide derivatives, and so on. However, Bestatin is the only marketed drug as APN inhibitor. Low activity and selectivety is the obstruction of APN inhibitors development. So it is necessary to exploit higher selective and more active APN inhibitors by rational design.According to the active site of APN, a good APN inhibitory should contain four parts. Part A:aromatic region which can act with S1pocket; Part B:hydrophobic amino acid side chain which can act with S1’ pochet; Part C:Zinc-Binding-Group (ZBG); Part D:a good linker. Based on our lab previous work, we designed and synthesized four series peptidomimetics compounds by modification of lead compounds15. In addition, preliminary APN and HDACs inhibitory assarys, in vitro anti-tumor cell proliferation assay and in vivo anti-tumor experiment were processed in order to find APN inhibitor with anti-tumor activity.We designed and synthesized four series of67target compounds and all the structures were indentified by1H-NMR and HRMS. All of these copmounds were novel without any report before.Preliminary enzyme activity assay showed that:In series one, the activity was decreased when the hydroxamic acid group was replaced by other ZBGs, which suggested that an approriate ZBG was essential to the activity. In series two, the linker of15replaced by other groups led to the bioactivity decreasing. However, we found the compounds containing ortho-substituted phenyl groups had better activities than those without substituents. In view of that, series three was designed. The enzymatic inhibition assay showed that ortho-substituent of phenyl group could greatly improve the activity of15, the IC50value of compounds3b and3c reaching to nanomole level. In series four, by introducing double-substituents in phenyl ring, we obtained compound4a which has the IC50values two order lower than Bestatin. The result of APN inhibition assay towards human ES-2cell surface was consistent with the result above, which suggested that our compounds had stong inhibition to human APN.The in vitro anti-tumor cell assay showed that target compounds had slightly inhibition against the growth of tumor cells, which was independent of the enzyme inhibition. It is according with our previous works and some articles. It’s reported that combination of5-Fu with APN inhibitors can improve the anti-tumor effect. Our results showed compounds4c can increase the anti-tumor effect of5-Fu. The in vivo anti-tumor experiment showed the same result, which may be due to inhibiting angiogenesis and cancer stem cell.Based on the new crystal structure of human APN and the interaction model of Bestatin with active sites, four series of67peptidomimetics compounds were designed and synthesized as APN inhibitors. The preliminary bioactivity assay showed compounds3c,4a which could be used as lead compounds for further anticancer agent research. The QASR and binding models of these compounds were also studied, which would be beneficial for the design and development of APN inhibitors in the future. |
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