In Vitro Activity Characterization of Novels Pyrazolo[3,4-d]Pyrimidine Prodrugs

The first part of this thesis focuses on the set-up of a bio-analytical and synthesis laboratory in line with ISO and GLP-Like requirements. Research projects carried out in the academic world while valid from a scientific point of view, are characterized by a poor level of accuracy and robustness due to the absence of a fine procedural and operational control. This defect inevitably compromises the quality of the data obtained, making the entire scientific project less attractive for investors such as pharmaceutical companies. Good Laboratory Practices (GLP) as well as ISO standards deal with the organization of processes and the conditions under which laboratory studies are planned, performed, monitored and recorded. GLPs are intended to promote the quality and validity of test data. The aim of the GLP is to promote the quality of "non-clinical" experimentation and the production of reliable experimental data to ensure the mutual recognition of the results obtained at an international level, reducing the costs for research. The main phase of setting up a certified laboratory was the drafting of detailed standard operating procedures (SOPs) which covered several topics such as instrumental use and maintenance, raw data recording and management, good laboratory practices, personnel responsibilities and analytical procedures, protocols and reports. Every single aspect of the laboratory activities starting from reagents used to instrument performance were recorded. The goal was not only the quality of the data, but also their traceability and integrity. Checking the documents produced it is possible to find information easily and unambiguously such as who did the study, how the experiment was carried out, which procedures were used, if there were any problems and how they were solved. In the second section, two novel Si113-prodrugs were synthetized introducing the amino acid sequence D-Ala-Leu-Lys (TP) to overcome the low water solubility of the compound. Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. Pyrazolo[3,4-d] pyrimidine derivatives, a promising class of Src family kinase (SFK) inhibitors have been extensively studied by Professor Maurizio Botta research group and collaborators. They exhibited strong antiproliferative and pro-apoptotic effects on several cancer cell lines. The prodrugs were fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The TP sequence introduced has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers including HCC and ovarian carcinoma. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. Preliminary tissue distribution and in vivo pharmacokinetic properties confirmed a better profile. Moreover, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (about 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.