Design, Synthesis And Preliminary Activity Assay Of Pyrrolidines MMPIs

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidase involved in the degradation and remodeling the extracellular matrix proteins. MMPs also play critical roles in many physiological processes, such as embryonic development, angiogenesis and wound healing. In normal physiological states, the activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), which mediate the stability of cells all together. The MMPs, which have been proved to play key roles in the processes of tumor growth, invasion, metastasis and angiogenesis, are frequently overexpressed in malignant tumors, and are associated with an aggressive malignant phenotype and poor prognosis in patients with cancer. Thus MMPs have become attractive targets for anticancer drug design. Accordingly, research on inhibitors targeting at MMPs, especially which have high selectivity at gelatinase (MMP-2, 9), is a very promising strategy in the development of current anti-angiogenesis therapy of tumor.Based on the 3D structures of known MMPIs and binding modes of these compounds in complex with MMPs, we designed 3 series of pyrrolidines compounds with the aid of Chem3D Ultra 7.0 program, which are N1-cinnamoyl substituted pyrolidine, N1-p-methoxyl cinnamoyl substituted pyrolidine and N1-caffeicoyl substituted pyrolidine. All the compounds designed were synthesized using 4-hydroxyproline as material through a reaction sequence including methylation, esterification, condensation, S_n2 substitution, acylation and etherification. Thestructures of target compounds were identified by IR, ESI-MS and HNMR spectra.Preliminary activity assay was carried out in vitro. Two kinds of enzymes were selected, which are gelatinase and APN. The inhibitory activities of these compounds against gelatinase (MMP-2 and -9) were measured using succinylated gelatin as substrate. After analyzing the structure-activity relationship of the pyrrolindine derivatives designed, we concluded that in some extent, longer and more flexible side chain meant higher activity against the enzymes. Furthermore, aromatic ring displacement could enhance the activities, and compounds with structure of butterfly had higher activities. In addition, APN is also a family of zinc-dependent metalloproteinase associated with malignant tumor, all of which have two zinc ions in the catalytic domain. In order to identify the selectivity to MMPs of the target compounds, the inhibitory activities of compounds on APN was also carried out with L-leucine /7-nitroanilide as substrate. As a result, most of these newly synthesized compounds also show inhibitory activities on APN. The results from the comparative studies exhibit that the selectivity towards MMP-2, 9 is higher than towards APN. According to the results of preliminary activity assay, it is stated that compound AO, A8, A10, B9, CIO and 4B have high inhibitory activities on MMP-2, -9, while compound 18B has good result on inhibiting APN. They are deserved to run further research in vivo which may become promising lead compounds in the future.In this thesis, we try to design the pyrrolidine derivatives that could conformationally match the requirement of the active site of MMPs, and had good inhibitory activity both on APN and matrix metallopeptidase-2, -9 in vitro. Some of these compounds, for example AO, A10, A8, B9 and CIO, are supposed to have potential anti-tumor activity in vivo and might be promising lead compounds, which would provide new structural backbones and directions for the development of a new generation of anti-tumor drugs.