4,6-Diphenyl-pyridines/pyrimidines and pyrazolo[3,4-d]pyrimidines: promising scaffolds as antiviral, anticancer and theranostic agents.

Nitrogen-based heterocyclic molecules received increasing attention in biological and chemical sciences, becoming a significant moiety in drug design. In this thesis, the research work has been focused on the design and the synthesis of novel derivatives based on the 4,6-diphenyl-pyridine/pyrimidine and pyrazolo[3,4-d]pyrimidine scaffolds as key pharmacophores for their antiviral and anticancer activities, respectively. The first part deals with the design and synthesis of a set of small molecules able to interfere with the Influenza (flu) RNA-dependent RNA Polymerase (RdRp) functions, exploiting the protein-protein interactions (PPIs) approach. An introduction of influenza disease and its pathogens is provided, focusing on the replicative mechanism of the influenza viruses and on the structural and functional information of the flu RdRp. The PPIs of the three polymerase subunits PA, PB1 and PB2, are reported and the inhibitors targeting the heterodimer PA-PB1 are introduced. Finally, the rational design and synthesis of new hybrid compounds bearing the 4,6-diphenyl-pyridine/pyrimidine cores are described togheter with the biological evaluation and molecular modeling studies. The second part of the dissertation focuses on the pyrazolo[3,4-d]pyrimidine compounds, on which both a lead optimization study and a theranostic design have been performed. The pyrazolo[3,4-d]pyrimidines have shown a promising activity both in in vitro and in in vivo as protein kinase inhibitors. The lead optimization study has been performed with the aim of obtaining a new class of derivatives as potent Src kinase inhibitors. The rational design, synthesis and biological analysis of the novel compounds are discussed. A further part of the project is dedicated to the design and development of potential theranostic prodrugs of two promising in-house pyrazolo[3,4-d]pyrimidines, SI306 and SI113. These prodrugs have been synthesized as potential agents for the diagnosis and the treatment of glioblastoma multiforme (GBM). The state of the art on theranostic applications of drugs, whose use is continually increasing, is also described

Exploring a New Generation of Pyrimidine and Pyridine Derivatives as Anti-Influenza Agents Targeting the Polymerase PA–PB1 Subunits Interaction

The limited range of available flu treatments due to virus mutations and drug resistance have prompted the search for new therapies. RNA-dependent RNA polymerase (RdRp) is a heterotrimeric complex of three subunits, i.e., polymerase acidic protein (PA) and polymerase basic proteins 1 and 2 (PB1 and PB2). It is widely recognized as one of the most promising anti-flu targets because of its critical role in influenza infection and high amino acid conservation. In particular, the disruption of RdRp complex assembly through protein-protein interaction (PPI) inhibition has emerged as a valuable strategy for discovering a new therapy. Our group previously identified the 3-cyano-4,6-diphenyl-pyridine core as a privileged scaffold for developing PA-PB1 PPI inhibitors. Encouraged by these findings, we synthesized a small library of pyridine and pyrimidine derivatives decorated with a thio-N-(m-tolyl)acetamide side chain (compounds 2a-n) or several amino acid groups (compounds 3a-n) at the C2 position. Interestingly, derivative 2d, characterized by a pyrimidine core and a phenyl and 4-chloro phenyl ring at the C4 and C6 positions, respectively, showed an IC50 value of 90.1 mu M in PA-PB1 ELISA, an EC50 value of 2.8 mu M in PRA, and a favorable cytotoxic profile, emerging as a significant breakthrough in the pursuit of new PPI inhibitors. A molecular modeling study was also completed as part of this project, allowing us to clarify the biological profile of these compounds

Identification and Biological Characterization of the Pyrazolo[3,4-d]pyrimidine Derivative SI388 Active as Src Inhibitor

Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4-d]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s. Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation

Synthetic Heterocyclic Derivatives as Kinase Inhibitors Tested for the Treatment of Neuroblastoma

none6In the last few years, small molecules endowed with different heterocyclic scaffolds have been developed as kinase inhibitors. Some of them are being tested at preclinical or clinical levels for the potential treatment of neuroblastoma (NB). This disease is the most common extracranial solid tumor in childhood and is responsible for 10% to 15% of pediatric cancer deaths. Despite the availability of some treatments, including the use of very toxic cytotoxic chemotherapeutic agents, high-risk (HR)-NB patients still have a poor prognosis and a survival rate below 50%. For these reasons, new pharmacological options are urgently needed. This review focuses on synthetic heterocyclic compounds published in the last five years, which showed at least some activity on this severe disease and act as kinase inhibitors. The specific mechanism of action, selectivity, and biological activity of these drug candidates are described, when established. Moreover, the most remarkable clinical trials are reported. Importantly, kinase inhibitors approved for other diseases have shown to be active and endowed with lower toxicity compared to conventional cytotoxic agents. The data collected in this article can be particularly useful for the researchers working in this area.openMusumeci, Francesca; Cianciusi, Annarita; D’Agostino, Ilaria; Grossi, Giancarlo; Carbone, Anna; Schenone, SilviaMusumeci, Francesca; Cianciusi, Annarita; D’Agostino, Ilaria; Grossi, Giancarlo; Carbone, Anna; Schenone, Silvi

Synthetic Heterocyclic Derivatives as Kinase Inhibitors Tested for the Treatment of Neuroblastoma

In the last few years, small molecules endowed with different heterocyclic scaffolds have been developed as kinase inhibitors. Some of them are being tested at preclinical or clinical levels for the potential treatment of neuroblastoma (NB). This disease is the most common extracranial solid tumor in childhood and is responsible for 10% to 15% of pediatric cancer deaths. Despite the availability of some treatments, including the use of very toxic cytotoxic chemotherapeutic agents, high-risk (HR)-NB patients still have a poor prognosis and a survival rate below 50%. For these reasons, new pharmacological options are urgently needed. This review focuses on synthetic heterocyclic compounds published in the last five years, which showed at least some activity on this severe disease and act as kinase inhibitors. The specific mechanism of action, selectivity, and biological activity of these drug candidates are described, when established. Moreover, the most remarkable clinical trials are reported. Importantly, kinase inhibitors approved for other diseases have shown to be active and endowed with lower toxicity compared to conventional cytotoxic agents. The data collected in this article can be particularly useful for the researchers working in this area.</jats:p

Early investigation of a novel SI306 theranostic prodrug for glioblastoma treatment

Glioblastoma multiforme (GBM) is one of the most aggressive malignancies with a high recurrence rate and poor prognosis. Theranostic, combining therapeutic and diagnostic approaches, arises as a successful strategy to improve patient outcomes through personalized medicine. Src is a non-receptor tyrosine kinase (nRTK) whose involvement in GBM has been extensively demonstrated. Our previous research highlighted the effectiveness of the pyrazolo[3,4-d]pyrimidine SI306 and its more soluble prodrug CMP1 as Src inhibitors both in in vitro and in vivo GBM models. In this scenario, we decided to develop a theranostic prodrug of SI306, ProSI-DOTA(Ga-68) 1, which was designed to target GBM cells after hydrolysis and follow-up on the disease's progression and improve the therapy's outcome. First, the corresponding nonradioactive prodrug 2 was tested to evaluate its ADME profile and biological activity. It showed good metabolic stability, no inhibition of CYP3A4, suboptimal aqueous solubility, and slight gastrointestinal and blood-brain barrier passive permeability. Compound 2 exhibited a drastic reduction of cell vitality after 72 h on two different GBM cell lines (GL261 and U87MG). Then, 2 was subjected to complexation with the radionuclide Gallium-68 to give ProSI-DOTA(Ga-68) 1. The cellular uptake of 1 was evaluated on GBM cells, highlighting a slight but significant time-dependent uptake. The data obtained from our preliminary studies reflect the physiochemical properties of 1. The use of an alternative route of administration, such as the intranasal route, could overcome the physiochemical limitations and enhance the pharmacokinetic properties of 1, paving the way for its future development

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/ Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/ Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents