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

Aminopeptidases N(APN,CD13), one of membrane-bound zinc-dependent exopeptidase, is widely distributed in mammalian tissues including kidney, intestine, liver as well as the central nervous system . 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. APN has been also proved to behave as a receptor for corona-viruses TEGV and 229E in humans. More impotantly, Saiko and coworkers have shown that APN (CD13) is involved in the regulation of tumour cell invasion and metastasis. Futhermore, APN is involved in processes of immunological regulation, a nd as a result, many immunologic active materials have been degradated and the ability of macrophage and NK cells to identify and kill tumor cells have been weakened.All these findings make this enzyme an interesting target for possible anti-tumor drugs research, which require the development of more potent and selective inhibitors.Based on the 3D structure and binging models of some APN inhibitors in complex with APN, two series of L-lynsine derivative are designed with the aid of Sybyl 7.0 program. The target compounds are synthesized using N⁶-[Cbz]-L-lysine as material through a reaction sequence including methylation, acylation, hydrolysis or aminolysis. The chemical structures of target compounds are identified by IR, ESI-MS and ¹H-NMR.Prelimi nary 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 against APN, and compound B6 is the best inhibitor with IC₅₀ value of 13.2μM, in contrast with the positive drug Bestatin, which IC50 is 15.5μM. Structure-activity relationships of twenty tested compounds are elucidated and compounds containing a hydroxamic acid as Zn2+ binding group have higher activity than carboxylic acid; With regard to R₁ group,compounds containing six-member aromatic heterocyclic have higher activity than five-member aromatic heterocyclic, and aromatic ring substituted by electron withdrawing group has better activity.To determine the selectivity of these inhibitors towards the targeted enzyme, inhibiting activity on MMP-2, which is one of endo-peptidases of Zn2+ metallopeptidases, are measured by using succinylated gelatin as substrate.Most of our compound show better inhibitory activity on MMP-2 than APN, and compound A2 is the best inhibitor with IC50 value of 2.93μM. One possible reason is that the catalytic center of MMP-2 is on the surface of the enzyme,however the catalytic center of APN is in the enzyme interior.Inhibitor molecule can reach the enzyme center of MMP-2 more easily.But compound B6, which is the best APN inhibitor of our 20 compounds, is the worst inhibitor of MMP-2 with IC₅₀ value of 170.0μM. We may come to a conclusion that compound B6 can inhibit APN selectively. Further pharmacological studies are performed on some compounds with high potency now.In this thesis, APN inhibitors of L-lysine derivatives have good inhibitory activity against APN and MMP-2 in vitro. One of these compounds, B6, who can inhibit APN intensively and selectively,is supposed to have potential anti-tumor activity in vivo and might be promising lead compound.