Design, Synthesis And Activity Study Of 1,3,4-thiadiazole Derivatives As APN Inhibitors

Partâ… .Recent Progress in Aminopeptidase NAminopeptidases N,one of membrane-bound zinc-dependent exopeptidase expressed mostly in intestine,is involved in the final hydrolysis of nutrients to amino acid,in the degradation of angiotensinâ…¢,in the decrease Na⁺-K⁺-ATP level,in the regulation absorption sodium in kidney,in the metabolism endogenous enkephalin and endorphin in the the central nervous system.APN,over-expressed on the surface of tumor cells,promots the invasion and metastasis of tumor cells via degrading extracellular matrix,promoting the release of most growth factor,stimulating the release of cellular factors associated with the neovascularization by vascular endotheilal cells,promoting the angiogenesis of tumor, speeding up the proliferation of tumor cells,enhancing the adhesiveness of tumor cells.APN is over-expressed on the surface of granulocyte and lymphocyte and participates the T-lymphocyte dependent inflammatory reaction.The enzyme is also over-expressed on the surface of antigen presenting cells,which degrades chemokines, antigen presenting molecule and lots of immunoactive substances,depresses the recognization of macrophage and NK cells to surface antigen on tumor cells and the ability to kill tumor cells directly,impairs the immunological functions.APN play a role in the entry of HIV-1 into host cells by degrading chemokine fMLP associated with the desensitization of the co-receptor CCR5 for HIV-1.APN serves as a receptor for human corona viruses HCoV-229E which causes upper respiratory infections and transmissible gastroenteritis viruses(TGEV) which causes a severe gastroenteritis in newborn pigs.Therefore,APN will be a good target for the design of novel therapeutic agents for the treatment of diseases,such as cancer,leukemia,rheumatoid arthritis,diabetic nephropathy and central nervous system diseases,such as Alzheimer's disease.APN inhibitors were designed to prevent and treat tumor,divided into two types, natural inhibitors and synthetic inhibitors.Most APN inhibitors thus far reported contain zinc binding group.Bestatin,firstly isolated from a culture filtrate of Streptomyces olivoreticuli,has been used in clinics as an immunoenhancer in Japan in 1987.Partâ…¡.Structure-based Rational Target Compounds DesignThe characteristics of this study are the utilization of structure-based drug design comprehensively.The structure information of the active site of E.Coli APN and enzyme-inhibitor complex was thorough studied with advantages of computer-aided drug design software.Flexsible Docking was used for modeling the interaction between target compounds and enzyme.APN exhibits a broad specificity for peptides with a N-terminal neutral or basic amino acid.The favored substrate of APN is alanine.On the other hand,the major components of ECM are Pro-Gly residues.So the alanine and the glycine were considered as the lead compounds.The 1,3,4-thiadiazole and the cinnamic acid which can interact with hydrophobic site of APN and has the property of anticancer was introduced into the target compounds. This scaffold is optimized with following chemical modification:(â…°) Various electron-attracting groups and electron-pushting groups was linked to target compounds so as to investigate the interaction of electric effect with APN;(â…±) The group which can form hydrogen bond with the enzyme was introduced to enhace affinity;(â…²) The group with various space volume was connected to target compounds to investigate the interaction of stereoscopic effect. Additionally,the matching of target compounds with the active site of enzyme is not only considered overall in this study,the fragments used in the target compounds also apply the fragments with anticancer activity and amino acid residual which can be compatible with tissues.The ADME and pharmacokinetics properties of the target compounds have also been considered in advance.Molecular weight, lipid-water partition,the number of hydrogen bond receptor and hydrogen bond donor fit the Lipinski rule and so increasing the hit rate.Partâ…¢.Synthesis and discussion of targeted compoundsOn the basis of structure-based drug design,we obtained 42 structural confirmed target compounds.The various substituted carboxylic acid was condensed with N-aminothiourea in presence of Phosphorus oxychloride,yielding 5-substituted-1,3,4-thiadiazol-2-amine.The coupling of amino acid with various substituted cinnamic acid led to amide using DCC.Finally,the intermediate 5-substituted-1,3,4-thiadiazol-2-amine and amide were reacted using DCC to give target compounds.The advantage of DCC method is mild reaction conditions and simple operation.All the targeted compounds are novel without any report by now, and the structures of new compounds are identified by IR,¹H-NMR,¹³C-NMR and ESI-MS.Partâ…£.The Biological EvaluationWe design and synthesis 42 novel 1,3,4-thiadiazole derivatives which were performed the in vitro preliminary activity evaluations.APN and MMP are zinc-dependent metalloproteinase and closely associated with the invasion and metastasis of tumor cells.So the in vitro enzyme inhibitory activity was determined against APN and MMP.The cells assay is performed toward leukemia cells(HL-60) which express APN.The preliminary test results showed that most of compounds have ability to inhibit APN.Six compounds,A7,A8,B4,B14,C5,C9 displayed good ability to inhibit APN with IC₅₀ values 31.4μM,68.7μM,35.3μM,27.1μM,44.6μM,41.1μM respectively.These compounds could be used as lead compounds for exploring novel APN inhibitors in the future.The results show that APN inhibition activity of compounds containing alanine acid residues is superior to MMP inhibition activity, while compounds containing glycine acid residues exhibited both APN and MMP inhibition activity.So the compounds containing alanine acid residues displayed selectivity toward APN.In in vitro growth inhibition against leukemia cells,the results indicated that most potent APN inhibitor(A7,B14,C5,C9) displayed good inhibitory effect against the growth of HL-60 cells.It is discovered that there is some relevance between part of the quantitative parameters(such as FlexX Docking score) and the in vitro activity of compounds.It shows that the study idea is rational.In conclusion,a series of novel 1,3,4- thiadiazole derivatives have been designed and synthesized.The preliminary inhibitory activities of target compounds were evaluated in this thesis.All of these discoveries provide a beneficial theoretical basis for the optimization of lead structure and lay a solid foundation for the discovery of novel more potent anticancer agents in future.