The Design, Synthesis And Preliminary Activity Assay In Vitro Of L-iso-Glutamine Derivatives As APN Inhibitors

Aminopeptidases N, one of membrane-bound zinc-dependent exopeptidase, is widely distributed in mammalian tissues including the central nervous system,the kidney and the intestine. In vivo, this enzyme is involved in the metabolism of angiotensin III in the brain and peripheral organs, in the degradation of nociceptin and in the inactivation of enkephalins, in association with neutral endopeptidase NEP. Furthermore, APN has been proved to be identical to a human lymphocyte surface cluster differentiation antigen CD13, and to behave as a receptor for corona-viruses TEGV and 229E in humans. APN has been also reported to play an important role in the invasion and metastasis of malignant tumor cells in vitro. All these findings make this enzyme an interesting target for possible anti-tumor drugs research, which require the development of potent and more selective inhibitors.Based on the 3D structures and binding models, two scries of L-iso-glutamine derivatives are designed with the aid of ChemOffice Program. All compounds are synthesized starting from the acylation reaction of ferulic acid with L-glutamic acid to form the basic scaffold, followed by the hydrolysis reaction in Na₂CO₃ solution and the key intermediate compound ((E)-2- [3-(3,4- dimethoxyphenyl) acrylamido] pentanedioic acid) was obtained. The intermediate compound is condensed withdifferent amino acids or primary amines, and produces the target compounds. The chemical structures of target compounds are identified by IR, ESI-MS and NMR ('H-NMR, I3C-NMR and HMBC) spectra.Preliminary bioactivity assays are carried out in vitro. Inhibitory activity of compounds against APN is measured with L-leucine p-nitroanilide as substrate. As a result, most of these newly synthesized compounds show good inhibitory activity on APN. Structure-activity relationships of tested compounds are elucidated and compounds containing aromatic heterocyclic substituents have higher activity than straight side chain; compounds substituted by halogen atom such as fluorine, chlorine and bromine had excellent inhibiting activity. Monoamide derivatives show higer activity than biamide derivatives. To determine the selectivity of these inhibitors towards the targeted enzyme, inhibiting activity on gelatinase (MMP-2, 9), which is one of endo-peptidases of Zn2+ metallopeptidases, are measured by using succinylated gelatin as substrate. Data of the comparative studies exhibits the selectivity towards APN is higher than towards MMP-2, 9.In this thesis, APN inhibitors of L-glutamate derivatives have good inhibiting activity both on APN and matrix metallopeptidase-2, 9 in vitro. Some of these compounds, for example AB01, are supposed to have potential anti-tumor activity in vivo and might be promising leading compounds.