Studies On The Design, Synthesis And Biological Activity Of The Integrin α_vβ₃ Antagonists

The growth of malignant tumors has been shown to be dependent on the development of new blood vessels. Prevascular stage tumors microfocus can produce a varity of diffusible angiogenic factors that induce host capillary endothelial cells to proliferate, migrate, and form new vessels in a process called tumor-induced angiogenesis. Once new vessels into tumor, The proliferate speed of tumor cells increases almost exponentially. Moreover angiogenesis provides also tumor cells metastatic passage. Therefore blocking tumor-induced angiogenesis is an efficiency strategy to prevent and cure cancer. It has been shown that the angiogenic process depends on vascular endothelial cell migration and invasion, processes regulated by cell adhesion receptor. The integrin αvβ3 is such a cell adhesion receptor. It has also be called as vitronectin receptor. The integrin αvβ3 receptor is a member of the integrin family of transmembrane heterodimeric glycoprotein complexs, which function in endothelial cell-cell and endothelial cell-extracellular matrix adhesion and communication. The integrin αvβ3 is expressed significantly on vascular cells in response to growth factors but is minimal on resting and normal blood vessels. Recent studies show the integrin αvβ3 and extracellular matrix can recognize each other through Arg-Gly-Asp (RGD) sequence, and that LM609 an antibody of integrin αvβ3 and some peptidescontaining RGD sequence antagonists can inhibit embryonic neovascularization, tumor-induced angiogenesis, and tumor growth. Now the SAR investigations of RGD-containing linear peptides and cyclic peptides show that the guanidinium of arginine and the carboxylate of aspartate is necessary for RGD recognition by integrin. NMR spectroscopic investigations of c(RGDfV), a highly selective αvβ3 ligand, indicate a more bent conformation of the RGD-sequence with ArgCα/AspCα distances of about 500 ppm and ArgCβ/AspCβ-distances of about 700 ppm fits better in the αvβ3 receptor than in the αvβ3 receptor, and a parallel arrangement of the side chains of arginine and aspartate. The turn type is not necessary for binding, but instead simply serves as a scaffolding for positioning the essential functional groups in proper juxtaposition. Soit provides opportunities to design nonpeptides antagonists of αvβ3 receptor. Recent studies of thalidomide shows that this severe teratological compound has distinct inhibition of angiogenesis and tumor metastasis. This mechanism maybe relate to down-regulate β3 integrin. The structure-activity investigations indicate the glutarimide ring of thalidomide is not necessary for antimetasis, but phthalimide is functional group.Taken together, based on these results, we use the lowest energy conformation of c(RGDfV) as template and thalidomide as lead structure for the development of peptidomimetics. Therefore three series target compounds derived from phthalimide were designed and synthesised. 1. The first series of target compounds contain glutamate or aspartate segment.T Ⅰ -01 ~T Ⅰ -07.2. The second series of target compounds contain β-alanine segment. TⅡ -01 ~T Ⅱ-10.3. The third series of target compounds contain β-phenyl-p-alanine segment.TⅢ-01 ~TⅢ-11.Such molecular design ideal and drug model of αv β3 antagonists using thalidomide as lead compound have not been reported in literatures.These target compounds can be classified two types from the synthetic method, such as 5-carboxy-1,3-dihydro-1,3-dioxo-2H-isoindole derivative and 5-amino-1,3-dihydro-1,3-dioxo-2H-isoindole derivative.The target compounds of 5-carboxy-1,3-dihydro-1,3-dioxo-2H-isoindole derivative were obtained starting from 5-isobenzofurancarboxylic acid via three different synthetic methods in 4 to 5 steps.The 5-amino-1,3-dihydro-1,3-dioxo-2H-isoindole derivatives of target compounds were obtained starting from phthalimide via 6 to 7 steps. In this paper detailed methodology of synthesis 4-nitro-N-carbethoxyphthalimide and the key intermediate of target compounds 5-amino-1,3-dihydro-1,3-dioxo-2H-isoindole benzyl ester were studies.Eighty-two intermediates were synthesised, among these forty-eight compounds have not been reported in literatures. Twenty-eight target compounds were obtained, and all have not been reported in literatures. These new compounds were confirmed by IR, 1HNMR, 13CNMR and MS.The biological activities of target compounds were studied by using endothelial cell proliferation assay, ADP-induced platelet aggregation and immune regulating model. The results indicated that most target compounds can inhibit ECV304 proliferation and be more active than RGDS tetrapeptide. Moreover it has been proved that the inhibition of endothelial cell proliferation is not the result of cytotoxic effect of target compounds. The ADP-induced platelet aggregation assay indicated that all target compounds have no effect on platelet aggregation. The results show that these compounds maybe not a ligend of α11bβ3. The results of immune regulating activity assay show that most target compounds have no effect on ConA stimulating T lymphocyte proliferation and LPS-induced B lymphocyte proliferation.The conformation of target compounds were analysed by using Silicon Graphics Iris Indigo II R4400 workstation. The studies show that Pharmacophore of target compound with highly activity were very similar to c(RGDfV).These researches established base for the searching of nonpeptide integrin αv β3 receptor antagonists.