The molecular basis of tumour angiogenesis has been extensively studied and the Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of this process. The various members of the VEFG family have overlapping abilities to interact with a set of tyrosine kinase receptors: VEGFR-1 and VEGFR-2 (KDR), largely expressed in endothelial cells and primarily involved in angiogenesis, and VEGFR-3, located in lymphatic vessels, where it seems to be critical in regulating lymphoangiogenesis.
The demonstration that expression of VEGF and of its receptors correlates with the degree of vascularization of many experimental and clinical tumours led to the rational design and development of agents targeting this pathway The new agents range from anti-VEGF monoclonal antibodies, such as bevacizumab, to small-molecule ATP-competitive VEGFR inhibitors, including compounds from distinct heterocyclic classes.
Inside the class of fused pyrimidines, those bearing the aniline substituted pyrimidine core showed to possess excellent antiangiogenic effects, and have widely been used as a basis for the design of new VEGFR-2 competitive inhibitors.
In this regard, my thesis work concerned the preparation of novel pyridothiopyrano-fused pyrimidines, characterized by an aniline group in the 2-position..
Moreover, with the aim to perform critical SAR studies, the new analogue 2-phenyl and 2-benzylamino derivatives were obtained.
The antiproliferative activity of new thiopyrano-fused pyrimidines was performed on HeLa (cervix adenocarcinoma), A-431 (epidermoid carcinoma) and MSTO-211H (biphasic mesothelioma) cell lines and the results are expressed as IC50 values, i.e. the concentration (M) of compound able to produce 50% cell death with respect to the control culture. All the anilino substituted derivatives show a notable antiproliferative effect, while both the addition of a methylene group and the deletion of the amino spacer abolished or drastically reduced the cytotoxicity. The obtained results clearly indicate a crucial role for the length of the side chain.
With the aim to investigate the mechanism of action responsible for the antiproliferative effect, the ability of new derivatives to inhibit the KDR tyrosine kinase activity was determined by a biochemical assay performed with a recombinant human kinase insert domain receptor.
The results, expressed as percentage of inhibition with respect to the control, clearly indicate for all anilino substituted derivatives a noteworthy capacity to inhibit the tyrosine kinase activity. Moreover, accordingly to the antiproliferative data, a significantly lower effect was induced by the 2-benzylamino or by the 2-phenyl analogues.
In particular, a comparison among the most active serie showed a notable effect for the p-methoxy substituted derivative, allowing some structure-activity relationships.