Synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine derivatives active as SGK1, Fyn and Src kinases inhibitors
Creators: name: GRECO, CHIARA
Publisher: name: Universit� degli studi di Genova
Year: 2020

description: The pathogenesis of many cancers is characterized by mutations, overexpression and dysregulation of protein kinases. As a result, increasing attention has been directed towards the identification of novel kinase inhibitors for cancer therapy. The work performed here focuses on the synthesis of a series of pyrazolo[3,4-d]pyrimidine derivatives as inhibitors of the serine-threonine kinase SGK1, and the tyrosine kinases Fyn and Src. The first set of compounds are analogues of the in house SGK1 inhibitor SI113 which had previously demonstrated anti-cancer activity. In fact SI113 resulted to be active on various cancer cell lines and in an in vivo hepatocellular carcinoma model. The new set of SI113 analogues are characterized by different anilines, ammines and a morpholine group in C4 and are decorated in C6 with polar chains, i.e. ethanolamine, diethanolamine, ethylene glycol and ethylenediamine. The double bond on the N1 side chain which is essential for the activity of compounds toward SGK1, was maintained. The structures of the second set of compounds, instead, are related to the in house Fyn inhibitor SI308. From previously studies on the first generation of Fyn inhibitors, SI308 was reported as the most potent compound, demonstrating both antiproliferative activity on cancer cell lines and the ability to inhibit protein Tau phosphorylation in a cellular model of Alzheimer?s disease. The new generation of SI308 related compounds present a methyl group at the C6 position and on C3 the phenyl ring is either unsubstituted or presents a methyl group in para position. Preliminary screening using enzyme activity assays demonstrates that some of the novel compounds are active and therefore suitable for further study using in vitro models. It is predicted that subsequent in vitro data will aid in the design of future compounds. Furthermore, previous data on another in house compound SI306, which is active on tyrosine kinase Src, reported promising results on an in vivo xenograft model of neuroblastoma. To further study this biological activity, SI306 was re- synthesized and additional experiments were performed on in vitro models. Testing of this compound on MYCN-amplified neuroblastoma cell lines HTLA-230 and SK-N-BE-2C further confirmed the activity of SI306 and provide increased support for the inhibition of Src as a valid approach for neuroblastoma treatment. Finally, further in vitro studies were performed on of the three previously cited in house pyrazolo[3,4-d]pyrimidines, SI306, SI308 and SI113, using patient derived glioblastoma multiforme (GBM) cell lines. This final set of work was undertaken during a visiting research fellowship period and performed in collaboration with the School of Pharmacy at the University of Nottingham (United Kingdom). Kinase inhibitor activity has been evaluated on series of patient derived GBM cell lines isolated from both the central tumor core (GCE28) and from the invasive margin of the tumor (GIN28 and GIN8). The use of such phenotypically relevant in vitro models represents an important step for GBM drug development and screening. The results gathered using these relevant cell models further demonstrate the anti-cancer activity of the pyrazolo[3,4-d]pyrimidines compounds. Moreover, investigating the compounds in combination with one another reveals that synergy can be achieved and this finding has additional implications for potentially overcoming GBM drug resistance. Additionally, to overcome the low water solubility of our pyrazolo[3,4-d]pyrimidines compounds, formulations of the lead compounds (SI306, SI308 and SI113) were prepared using miniaturized screening process based on inkjet printing technology6. The observed activity of our compounds in vitro taken together with their successful formulation highlight that our kinase inhibitors are attractive candidates for the treatment of GBM.