Design, synthesis and biological evaluation of heterocyclic compounds as potential anticancer agents

This doctoral thesis is the final product of the Ph.D. program in Sciences and Technologies of Chemistry and Materials, curriculum of Pharmaceutical, Food, and Cosmetic Sciences at the Department of Chemistry and Industrial Chemistry (DCCI) of the University of Genoa. The synthetic work was conducted at the Department of Pharmacy under the supervision of Prof. Silvia Schenone and at the Department of Drug Design and Pharmacology at the University of Copenhagen under the supervision of Prof. Bente Frølund. The computational studies presented in this thesis were carried out by Prof. Elena Cichero (University of Genoa) and Dr. Francesco Bavo (University of Copenhagen). In vitro and biological data were collected by Prof. Santina Bruzzone (University of Genoa), Dr. Vago (San Raffaele Institute in Milan), Dr. Previtali (University of Copenhagen) and Prof. Bogdanov’s group (University of Southampton). The thesis is organized into three chapters, each corresponding to one of the research lines that have defined the following doctoral activity over the past three years: 1. Structure-based design of pyrazolo[3,4-d]pyrimidines as SIRT2 inhibitors and their molecular simplification to diamino-1,3,5-triazines. 2. Pyrazolo[3,4-d]pyrimidine derivatives as potential anticancer agents for prostate and bladder cancer: SAR expansion, halloysite nanotube conjugation, and Src inhibition profile. 3. Development of GQX analogues coupled with a ROS-sensitive moiety to afford ROS-responsive prodrugs active on GABA receptors. Chapters 1 and 2 focus on pyrazolo[3,4-d]pyrimidine derivatives, since their scaffold constitutes a privileged chemotype in medicinal chemistry, functioning as an isostere of the adenine ring in ATP, enabling it to emulate the binding interactions at the kinase active site hinge region. This scaffold possesses substantial pharmacological and biological significance, including notable potential as an anticancer agent. The synthesis of amino-substituted pyrazolo[3,4-d]pyrimidines SIRT2 inhibitors, guided by structure-based design, and its molecular simplification into diamino-1,3,5-triazines, is discussed in Chapter 1. Moreover, in Chapter 2 pyrazolo[3,4-d]pyrimidine derivatives were also explored for potential applications in uro-genital cancer. Diversifying the substituents, a broader structure-activity relationship (SAR) analysis was performed, also enabling both covalent and adsorptive attachment to halloysite nanotubes, a delivery system aimed at improving solubility and therapeutic administration. Current research efforts include Src kinase inhibition profiling, chemical stability evaluation, and studies of carrier conjugation to elucidate factors influencing cellular efficacy and to inform the selection of lead candidates. Motivated by the emerging evidence for an immunosuppressive role of GABA signalling within the tumour microenvironment, Chapter 3 focuses on the synthesis of GQX (guanidinium–quinoxaline) antagonists, followed by their conversion into ROS-responsive prodrugs through installation of a boron-containing, oxidatively cleavable promoiety. This prodrug strategy is intended to enable site-selective unmasking in ROS-enriched malignant tissues, thereby coupling on-target GABA-receptor antagonism with improved delivery characteristics. Taken together, the development of diversity-oriented routes to pyrazolo[3,4-d]pyrimidines, 1,3,5-triazines, and GQX-based chemotypes yielded previously unreported notable compounds spanning SIRT2 inhibition, c-Src–associated antiproliferative activity, and ROS-triggered GABA-receptor antagonism. These results substantiate the pharmaceutical promise of these heterocyclic frameworks and delineate a coherent platform for subsequent optimization, translational delivery studies, and mechanism-driven biological assessment in oncology-relevant settings.