Design, Synthesis And Anti-tumor Activity Study Of Ureido Peptidomimetics As Aminopeptidase N Inhibitors

Part I. Research Backgroud of Aminopeptidase NAminopeptidase N (APN/CD13) is a zinc-dependent metalloprotease and a membrane binding exopeptidase belonging to Ml family, which transmembrane-binds as an integrin dimer on cell surface. According to tumor cell biology, APN is observed to be highly expressed by various mammalian tumors, and plays significant roles in tumor proliferation and metastasis as a protein hydrolyase during tumor invasion, metastasis and angiogenesis process. It is reported that the roles of APN in tumor includes:(1) degradation of extra cellular matrix (ECM), to directly promote tumor cell invasion and metastasis; (2) degradation of ECM, resulting to the release of various growth factors, facilate tumor proliferation; (3) induce of vascular endothelial cells releasing cell factors related to angiogenesis, to promote the formation of capilliary tubular structure.Angiogenesis is a complex process, only observed in newborns, adult callus or mammaliam tumor. When the diameter of primary or secondary tumor mass was more than 1-2 mm3, new vessels must be developed to support nutrient; tumor metastasis can also induce angiogenesis. Angiogenesis includes endothelial cells to invade into the basement membrane, migration, adhension and proliferation, and to form net structure in tumor mass finally. ECM mainly consists of collagen, proglygan, fibronectin and laminin, etc, to form fiber-like net complex with collagen and proglygan as main scaffold, which is important to cell connection and signal transduction. It is the main barrier of tumor metastasis, therefore, degradion and invasion of ECM is the first step of tumor metastasis and angiogenesis.It becomes more and more concentrated on APN targeted anti-metastasis and anti-angiogenesis agents because of the tight relationship between APN and tumor proliferation, invasion, metastasis and angiogenesis. Among reported APN inhibitors, Bestatin from Streptomyces ofivorecticuli as a natural product immune regulator is in market in Japan in 1987. So far, researchers still concentrate on its roles in tumor metastasis and angiogenesis at high concentration and some progress have been obtained.PartⅡ. Rational Design and Synthesis of Target CompoundsAccording to peptidomimetic design strategies, we use ureido group to replace amide bond (peptide bond) commonly seen in natural peptides as the linker, and non-peptide substitutes to replace amino acid residues as improved pharmacophore, which is linking to neutral or basic amino acid residues preferred to by APN, to form small-molecule ureido peptidomimetic compounds. We make modification and improvement to the compounds, including the rational design of the linkers and the side chains accroding to virtual docking and bioactivity screening, and successfully obtained them by directed synthesis.The compounds designed in this paper obtained extremly obvious improvement of their activity after rounds of modification. And the several most potent ones were studied in further in vitro anti-tumor mechanism and in vivo pharmacodynamics mainly on anti-metastasis and anti-angiogenesis. The results obtained were beneficial to further modification of APN inhibitor scaffold and pharmacophore and the construction of complete in vitro and in vivo bioactivity evaluation models for APN inhibitors. The compounds designed in this paper were potential to be developed further.Based on virtual docking and rational design, we modified the compounds on their side chains, non-peptide substitutents and linkers to enrich their structural diversity. Seventy eight target compounds were obtained by directed synthesis. The advantage of peptidomimetic synthesis can be seen in the synthetic process, in which the schemes can be completed with high efficiency and yields.The three series of target compounds can be synthesized through two schemes:(1) In order to study the influence of the amino acid side chains and non-peptide substitutes on the bioactivity, with various aliphatic or aromatic amines as start materials, they were transformed into related isocyanates, then coupled with various amino acid methyl esters to form ureido groups, and finally the methyl ester was transformed into hydroxamic acid, which would be the target compounds. (2) In order to study the influence of the linker or the amino acid side chains and ZBG on the bioactivity, with various amino acid methyl esters as start materials, they were transformed into related isocyanates, then coupled with amino acid methyl esters or alcohols, and finally the methyl ester was transformed into various ZBGs. The structures of the target compounds were comfirmed with 1H-NMR,13C-NMR and HRMS. Their purity was comfirmed with element analysis and HPLC, above 95%. Through reference, we can see that the target compounds are novel chemical entities, which has never been reported yet.PartⅢ. The Bioactivity Evaluation, in vitro Mechanism and in vivo Pharmacodynamics Studies of the Target CompoundsIn this paper, three series of 82 small-molecule peptidomimetics were obtained, and they were assay towards APN from porcine kidney as a primary screening, in which the ones with higher enzyme inhibitory activities would be further studied, including of their enzyme inhibitory activities towards APN expressed on B16BL6 mouse melanoma cell and ES-2 human ovarian clear cell carcinoma cell surfaces, their influen on the survival of the two cell lines, and their selectivity over MMP-2 which is also a zinc-dependent metalloprotease responsible for ECM degradation. The most potent and selective APN inhibitors with the least influen on the two tumor cell line survival would be further studied on their anti-invasion and anti-angiogenesis mechanism in vitro. According to the in vitro results, the most potent ones would be selected to be assay for their anti-metastasis and anti-angiogenesis pharmacodynamics in vivo further.The enzyme inhibition assay showed that ZBG is essential to the bioactivity, as well as the linker, the amino acid side chains and the non-peptide substitutes. Half of the 78 compounds showed to be more potent than Bestatin the positive control, among which more than ten ones has their IC50 values one order of magnitute lower than Bestatin. It demonstrated that the ureido peptidomimetics as small-molecule APN inhbitors were quite potential. Otherwise, the most potent ones has high selectivity over MMP-2, and their GI50 values were more than Bestatin which has been in the market according to the MTT assay.In vitro mechanism studies consist of two parts, anti-invasion and anti-angiogenesis. In the anti-invasion assay, transwell chambers coating with Matrigel were adopted. In the anti-angiogenesis assay, rat aortic ring microvessel growth model and HUVEC tubular structure formation model were adopted, in which Matrigel was also needed. Matrigel was a commertiallized product secreted by mouse EHS sarcoma as its ECM, which was used to replace human ECM in the laboratory. The results of the anti-invasion assay showed that the ureido peptidomimetics 3-11, 3-19,3-36,3-40,3-34, and 3-45 can significantly block ES-2 cell invasion without obvious influence on ES-2 cell migration. The results of rat aortic ring microvessel growth and HUVEC tubular structure formation assay showed that the ureido peptidomimetics 3-11,3-19,3-36,3-40, and 3-45 had significant in vitro anti-angiogenesis effects.The in vivo anti-metastasis and anti-angiogenesis pharmacodynamics studies of the ureido peptidomimetics consist of two models as experimental lung metastasis and tumor intradermal tranplant-induced angiogenesis. In both of the two models C57BL/6 mouse and B16BL6 mouse melanoma cell line were adopted. The in vivo results showed that the ureido peptidomimetics 3-36,3-40, and 3-45 can significantly inhibit the metastatic pulmonary nodule formation. They can also significantly inhibit new vessels formation induced by the intradermal tumor.The three compounds selected in the in vivo assays performed to be in consistent with their enzyme inhibitory activities and to be more potent the Bestatin the positive control, demonstrating that they would be developed as anti-tumor candidates.PartⅣ. The Structure-Activity Relationship Studies of the Target CompoundsWe selected three representive compounds screened from the enzyme inhibitory activity assay towards APN, to be investigated for their binding modes with the target using virtual docking, in order to direct further compound design and modification. The molecular docking applied APN from E.coli (eAPN) as the receptor, with the condition in which Bestatin can be restored as its primary conformation of the complex. The results showed that compound 1-18 and 3-11 adopted similar binding modes to Bestatin, but the 1-naphthylmethyl substitute of compound 3-45 stretched towards a different subsite. We speculated that it may be the reason for the good enzyme inhibitory activities of 1-18 and 3-11 and the breakthrough of 3-45 towards APN.PartⅤ. ConclusionThe ureido peptidomimetic small-molecule APN inhibitors studied in this paper have potent APN inhibitory activities. We mainly concentrated on the physiological and pathological funtion of APN on ECM degradation as tumor cell surface marker, which plays important roles in tumor metastasis and tumor-induced angiogenesis. The models were successfully constructed and significant anti-tumor results were obtained. The in vitro and in vivo results showed that the ureido peptidomimetics has the potential to be modified as lead compounds and anti-metastasis as well as anti-angiogenesis candidates with clinical values.