DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF POTENTIAL STAT3 INHIBITORS AS ANTICANCER AGENTS

Signal Transducers and Activators of Transcription factors (STATs) are a class of latent cytoplasmic proteins that regulate cell growth and survival by modulating the expression of specific target genes. One member of the STAT family, STAT3, has received particular attention since it has been found constitutively activated in a broad spectrum of cancer cell lines and human tumors. These compelling results, combined with a well-known selectivity of STAT3 inhibitors for tumor cells, validated STAT3 as a promising anticancer drug target. Although the experimental studies on STAT3 inhibitors seem very interesting, to date, there is no evidence in literature of any impending clinical development. The aim of this research project was the identification of new small molecules able to inhibit STAT3 activity through the direct binding to STAT3 protein. Starting from the structures of molecules which were known in the literature for their STAT3 inhibitory activity, three classes of compounds were developed: the oxadiazoles, the pyridazinones and the ortho-quinone derivatives. The oxadiazoles were structurally related to AVS-0288, which was identified through a screening of a Korean chemical library for its ability to inhibit STAT3 activity, although the exact mechanism of action had not been clarified yet. These derivatives were designed using the most classical approaches for a ‘hit to lead generation’: bioisosterism, vinilogy, homology. The second series of molecules, the pyridazinones, derived from the natural compound Cryptotanshinone, a direct STAT3 inhibitor, and was designed with the support of molecular modeling studies. These latter studies suggested a structural similarity between Cryptotanshinone and a series of pyridazinone derivatives, previously investigated by our research group. Moreover, in order to combine the structural characteristics of the two series of molecules above, chimeric compounds, on the basis of an accurate conformational analysis, were designed and synthesized. Finally, with the aim to understand the mode of interaction of the parent compound Cryptotanshinone with the SH2 domain of STAT3, docking studies and in vitro screening were carried out. These suggested a key role of the ortho-quinone moiety, since it resembled the phosphotyrosine of the natural peptide able to bind SH2 domain. Therefore, ortho-quinone compounds, derived from a simplification of Cryptotanshinone structure, were synthesized. However, due to the instability and high volatility of some of the designed compounds, only two representative terms have been prepared to date. All synthesized molecules were evaluated by a cell-based preliminary screening, the dual-luciferase assay, which selected for compounds able to exert an inhibition on STAT3 activity. Although some of these assays showed interesting preliminary results, a deeper investigation was fundamental, in order to determine their target and/or mechanism of action. With this aim, an in vitro competitive binding assay, the AlphaScreen technology system, was performed to check the ability of these compounds to bind the SH2 domain of STAT3 and inhibit its dimerization, a crucial step for STAT3 activation. This biological investigation led to the identification of the oxadiazole amidic derivative F2e, which was found to exert positive results both in the luciferase assay (20% inhibition at 5 μM) and in the AlphaScreen-based assay (IC50 = 17.7 μM), and moreover, its inhibitory activity was found to be dose-dependent. The effect of F2e on the tumor cell growth was also checked and shown to have a good profile of inhibitory activity on cell proliferation, with an GI50 around 2 μM for most of the screened tumor cell lines. Due to these encouraging results, F2e is considered the lead compound for the development of a new series of derivatives.