| Objective:Aminopeptidase N (APN/CD13; EC 3.4.11.2), a zinc-dependent membrane metalloproteinase, is present in a wide variety of human tissues and the surface of cell types (endothelial, epithelial, fibroblast, leukocyte). Compared with normal cells, APN is over-expressed on the surface of some tumor cells. It is demonstrated that this enzyme plays a crucial role in the process of tumor growth, invasion, metastasis and promoting the angiogenesis. Therefore, APN is considered as an important target in anti-cancer drug discovery and development.So far, many natural inhibitors such as Bestatin, Phebestin, Probestatin, Amastatin, Curcumin have been reported and proved to be clinically efficacious for the treatment of tumor. Bestatin, the first marketed drug as natural aminopeptidase N inhibitor in Japan in 1987, now is clinically used to prolong the survival of patients with acute adult nonlymphcytic leukemia. In recent years, numerous synthetic APN inhibitors(APNI) have been reported, such asα-aminophosphonates APNI, a-aminoboronic acid APNI,β-thiols APNI,2,6-piperidinedione-N-acetamide derivatives APNI, L-lysine derivatives APNI, AHPA (β-Amino-α-Hydroxyl-phenylbutanoic acid) derivatives APNI and so on.In this study, we designe and synthesize a series of small molecule peptido-mimetics based on the APN as target. Their preliminary biological activity screening are subsequently conducted in order to find out effective APN inhibitors as anti-cancer lead compounds.Methods:According to the binding characteristics learned from the three dimensional crystal structure of APN and the mode of action of APN-inhibitor complex reported in literatures, we choose the chloramphenicol amine as the novel scaffold based on the active structure of 3-phenylpropane-1,2-diamine scaffold which was previous reported by our group. To improve the interaction with the hydrophobic pockets in the active site of APN, several fragments were introduced to the scaffold. This scaffold is optimized with following chemical modification:i) The different space volume and length of side chains were chosen to investigate the interaction of stereoscopic effect with APN. ii) Various electron-attracting groups and electron-pushing groups were introduced so as to investigate the interaction of electric effect with APN. iii) The groups which can form hydrogen bond with the enzyme were taken to enhance affinity. All the target compounds were rationally designed by CADD, FlexX docking and virtual screening. Meantime, the properties of all these compounds like molecular weight, lipid-water partition, the number of hydrogen bond receptor and hydrogen bond donor were predicted and screened by Lipinski rules before synthesis.115 novel target compounds were designed and synthesized by using chloramphenicol amine as starting material, through amino protection, selective oxidation, bromination, azide, reduction, condensation and other reaction steps. The structure were confirmed by IR,1H NMR, ESI-MS, X-ray crystal diffraction methods.In addition, enzyme assay (APN, MMP-2), cancer cell (HL-60, ES-2, K562, A549, H7402,2PLC) proliferation assay and the mice experiment in vivo were processed in this research.Results:In this study 115 target compounds were synthesized and most of the compounds were obtained in high yield and good purity. All of the compounds were novel without any relevant reports in literature.In series A, the preliminary activity evaluation against APN showed that some compounds possessed moderate inhibitory activities. Among 78 target compounds, 9e(9e'),9m(9m'),9r(9r') and 9s(9s') were the most potential. Compared with series A,25 compounds in series B exhibited better inhibitory activities. The IC50 of compounds 12w,12x and 12y were below 35μM, which were close to that of the positive control Bestatin. However, the high inhibition of MMP-2 was also observed. In series C, compound 161 was found to be the most active. The inhibitory activity was similar to that of Bestatin. Generally speaking, compounds in series C showed low activities towards MMP-2. It is worth mentioning that compounds 16k and 161 not only had a high inhibition against APN, but also represented low inhibition towards MMP-2, which suggested that both the compound have good selectivity against enzymes.The structure activity relationship (SAR) was summarized based on the test results of the target compounds with the scaffold chloramphenicol amine in the previous enzyme assay. Comparative Molecular Field Analysis (CoFAR) was utilized to establish the QASR model of target compounds, which showed good cross-validated coefficient q2 and predictive potency. According to the the steric contour map and the electrostatic contour map of the CoMFA model, a program on the further structural optimization was proposed.The test results of growth inhibition against six tumor cells in vitro indicated that most potent APN inhibitors displayed good inhibitory effect against the growth of these tumor cells. Compound 161 exhibited very similar potency against the proliferation of tumor cells compared with that of Bestatin. Compound 12w showed better inhibitory effect towards all the tumor cells than Bestatin.In vivo experiments, compound 12w and 161 showed high potency against tumor cell H22 metastasis with the inhibition rates 48.51% and 58.04% respectively in mice. While the inhibition rate for the positive control Bestatin was 56.49%.Conclusions:In conclusion, based on CADD and the virtual screening with APN as target chloramphenicol amine derivatives were designed and synthesized as APN inhibitors. We reported a convenient and economical method of the synthesis of APN inhibitors. Preliminary activity assays showed that most compounds displayed good APN inhibitory effects. Some compound such as 12w,161 showed better activities than the positive control Bestatin and could be used as lead compounds in the future. We also established a QASR model of target compounds, which is beneficial for the design of novel APN inhibitor in the further study. |
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