• A2B
• benzothiopiranoindole
• DNA Topoisomerases
• dual inhibitors
• indazoloquinazoline
• indole-based compounds
• non-campthotecin derivatives
• P2X7
• PET imaging
• purinergic system
• purines
• pyridothiopiranoindole
• pyrimidines
• Topo I
• Topo II
• Tyrosyl-DNA phosphodiesterases

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) mediate diverse biological effects via two main families of purine receptors: P1 and P2 receptors. Adenosine/P1 receptors have been further subdivided, according to convergent molecular, biochemical, and pharmacological evidences into four subtypes, A1, A2A, A2B, and A3, all coupled to G proteins. Based on differences in molecular structure and signal transduction mechanisms, P2 receptors divide naturally into two families of ligand-gated ion channels and G protein-coupled receptors termed P2X and P2Y receptors, respectively; to date, seven mammalian P2X receptors (P2X1–7) and at least eight mammalian P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14) have been cloned, characterized, and accepted as valid members of the P2 receptor family. Extracellular purines and pyrimidines have important and diverse effects on many biological processes including smooth muscle contraction, neurotransmission, exocrine and endocrine secretion, immune response, inflammation, platelet aggregation, pain, and modulation of cardiac function. Additional studies have shown the role of purines in emergency situations, such as cerebral and myocardial infarct, epileptic seizures, and infections where these molecules serve as danger signals family. During the last decade, PET has become a valuable tool in the development of new drugs. Non‐invasive imaging using PET would allow studying biological targets in both healthy and diseased condition. Furthermore, it would be very useful in the drug development process since it gives direct insight in the relation between receptor occupancy and the dose of the candidate antagonist, allowing to validate or invalidate a new drug candidate at an early stage, thereby saving a lot of effort and money, making the drug development process more efficient.
In this view, we planned the synthesis of new PET radiotracers towards purinergic system, focusing our attention against A2B and P2X7 receptor subtypes.
A2B adenosine receptor is a G-protein-coupled receptor and it activates the cAMP-dependent pathway, by activating adenylyl cyclase through action of the Gs alpha subunit, and the phosphatidylinositol pathway, by activating phospholipase C through Gq subunit. It determines an enhancement in intracellular level of calcium, activation of IL6 pathway and NO synthesis. Due to its involvement in several physiopathological conditions, including angiogenesis induction, myocardial ischemia, kidney and lung injury, tumors, glucose metabolism, and osteoblast differentiation, A2B AR represents a valuable therapeutic and diagnostic target for different diseases, such as diabetes, tumours, cardiovascular diseases, pulmonary fibrosis and others. Anyway, the limited availability of potent and selective ligands has prevented an inner characterization of the receptor for years.
We have recently studied the class of 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones in search for A2B AR ligands with high affinity and selectivity. Actually, a number of new derivatives was disclosed that resulted completely inactive and moderately active at A3 and A1 ARs, respectively, whereas showed a A2B AR/A2A AR selectivity degree strictly dependent on the aryl group (Ar) at the 3-position of the central core. On the basis on these results, we investigated the aryltriazinobenzimidazole scaffold, in order to develop PET radiotracers as useful tools to deeper study and characterize A2B ARs.
In collaboration with Dr. Menichetti and his collaborators of CNR, Pisa, our designed compound was synthesized in high radiochemical yield and tested by PET to evaluate its pharmacokinetics in vivo, and to ascertain its potential use for A2B AR imaging. The molecule showed a very high chemical stability in saline and in plasma, and a good pharmacokinetic profile. Results of in vivo and ex vivo studies, mRNA and RT-PCR are in agreement, and showed the ability of this molecule to bind the A2B AR. Although further studies are required to better characterize this probe, our compound may represent a good lead compound for the development of new A2B AR radiotracers with improved selectivity and potency of binding. These results have been published on Nuclear Medicine and Biology, 43 (2016) 309–317.
During my PhD, I spent a research period in the Molecular Imaging Branch Laboratory, National Institute of Mental Health, National Institute of Health, Bethesda, Maryland, USA with Dr. V. W. Pike and Dr. M. Haskali. This visit was part of an ongoing collaboration between University of Pisa and MIB, aimed at developing new radiotracers for imaging brain P2X7 receptor in neuroinflammatory conditions with positron emission tomography (PET).
The P2X7 receptor (P2X7R) has an important role in inflammation and immunity, since its stimulation by endogenous level of ATP is correlated to the pro-inflammatory cascade culminating in activation of Interleukin-8 (IL-8), an inflammatory cytokine. More specifically, P2X7R is a ionotropic receptor activated that require extracellular concentrations of ATP in the range of 1 mM, in contrast to concentrations of ≤100 μM needed to activate other P2 receptors. The ATP molecule binds to and activates P2X7, resulting in pore formation. This leads to K+ efflux from the cell, which is a crucial step in inflammasome assembly. Prolonged activation of the P2X7R results in irreversible pore formation and allows the non-selective passage of ions and hydrophilic solutes of up to 900Da; this can result in colloido-osmotic lysis and cell death by apoptosis or necrosis. Pore formation is also thought to allow entry of bacterial products and extracellular ATP into the cell, which drives inflammasome formation.
P2X7R is also involved in neuroinflammation since it affects microglial cells, which are the primary immune cells of the CNS. Microglia play an important part in the immune system of the CNS by acting as scavenger cells. Activation of P2X7R by ATP in microglial cells results in the release of autolysosomes into the extracellular space, providing a mechanism for the clearance of intracellular pathogens. P2X7R also plays a role in the generation of superoxide in microglia. Thus, P2X7R has been implicated in the pathophysiology of Alzheimer’s disease and other neurodegenerative conditions through ATP-mediated cortical cell death and superoxide release.
As far as we know, the only molecule for this purpose reported in literature being able to cross the BBB in rats and showing good results both in rats and monkeys is 11C‐JNJ‐54173717. In developing a new PET radiotracer, we chose a molecule already patented as antagonist which shown an IC50 value of 2 nM and a pIC50 value of 8.64 M. The product was obtained and characterized but due to technical issues we weren’t able to perform the last radiosynthtetic step and the project is still ongoing.
In parallel to these diagnostic applications, I focused my attention on design and synthesis of novel anticancer compounds. Cancer is an increasing worldwide emergency and its incidence and mortality will double in the next twenty years, so it seems to be clear that we need new and more effective pharmacological therapies. Our attention was focused on DNA Topoisomerases: a class of enzymes that are responsible for solving complex topological problems of the DNA. They prevent excessive supercoils which may cause functional and structural alteration in cells. The mechanism of action consists in catalyzing the break of DNA's phosphodiester skeleton through the formation of a transient cleavage complex with the nucleic acid. Topoisomerase I catalyzes a single-stranded break, while Topoisomerase II a double-stranded one. After the cut, these enzymes catalyze the reanniling of segments. The formation of cleavage complex is due to the presence of a tyrosine residue conserved in all classes of topoisomerases and this intermediate represents the target of inhibitors.
Firstly, we started with the development of non-CPT derivatives against Topo I. Today there are two drugs approved for therapy, Topotecan and Irinotecan, which are Campthotecin derivatives. Campthotecin is a cytotoxic quinoline alkaloid which is extracted from the bark and stem of Camptotheca acuminata with remarkable anticancer activity but also low solubility and high reverse drug reaction. The new derivatives have partly solved these negative effects, but they maintained the lactonic ring responsible for chemical instability and part of the toxicity. So, research has moved towards design and synthesis of new classes of non-CPT derivatives like indolocarbazoles, phenanthridines and indenoisoquinolines. Recently our research group published a new series of compounds based on phenylpyrazoloquinazoline structure, which mimics the central core of phenanthridines and indenoisoquinolines.
From biological assays, four structures demonstrated the best activity against Topoisomerase I. They have been subjected to docking studies performed by the research team of Professor Novellino from University of Naples in order to rationalize the obtained results.
Based on these structures we design a new series of potential Topo I inhibitors featuring a new scaffold, in which the pendant phenyl of phenylpyrazoloquinazolines is fused with the other rings to constitute an indazoloquinazoline core. At position 5 a dialkylaminoalkyl chain is present with an NH- or O-linker.
Preliminary results on N-linked compounds showed that they are inactive against Topo I, while O-linked compounds are still under biological evaluation at Professor Pommier’s laboratory, Center for Cancer Research, National Institute of Health (NIH), Bethesda, MD (USA).
Considering its crucial role in such important processes Topo II enzyme has been widely exploited for cancer therapy. Drugs targeting Topo II include both poisons, which comprise most of the clinically active agents, such as etoposide, and pure inhibitors, such as the anthracycline aclarubicin.
Despite their efficiency in the clinic, current anticancer therapies with topoisomerase-directed agents are limited by some important negative consequences, with the most important ones arising from the observation that treatment with Topo II targeting drugs may result in secondary malignancies. In addition, the emergence of drug-resistant tumor cells remains one of the major problems, and is a frequent cause of failure in long-term clinical therapies. In this context, it has long been suggested that such resistance may be overcome, at least in part, by the ability of drugs to target both Topo I and Topo II simultaneously. In addition, Topo I and Topo II have overlapping functions in DNA metabolism, so that targeting both enzymes might increase overall anti-tumor activity.
The research unit I’m working with, has extensively studied several polycyclic chromophores, structurally related to classes of DNA intercalating agents, that exhibited the ability to intercalate with DNA and in some cases to inhibit topoisomerases I/II. Among them were synthesized compounds incorporating the purine, benzimidazole, and indole moieties, and more recently it was developed an extensive study on the series of benzothiopyranoindole derivatives.
Biological results of benzothiopyranoindoles showed an antiproliferative activity at low micromolar concentrations on HeLa (cervix adenocarcinoma) and HL-60 (promyelocytic leukemia) human tumor cell lines. The presence of a basic side chain (dialkylaminoalkyl) inserted at the 11-position on the indole nitrogen, seemed to be required for the cytotoxicity.
The successful results from the described benzothiopyranoindoles prompted the synthesis of novel derivatives. Pyridothiopyranoindole scaffold was chosen in order to expand structure-activity relationship knowledge. In this case, the introduction into the chromophore of a protonable nitrogen atom could provide an additional or alternative anchor point in the formation of the intercalation complex. All derivatives were functionalized with dialkylaminoalkyl chains, considering their crucial role for the biological activity.
The biological evaluation of all compounds synthesized in this thesis was conducted in collaboration with a research group of the Faculty of Pharmacy, University of Padua. The antiproliferative activity is usually tested in vitro on human tumor cell lines, representative for different types of tissue: HeLa (cervical adenocarcinoma), A-431 (squamous cell carcinoma) and MSTO-211H (biphasic mesothelioma). All tested compounds exert a significant antiproliferative activity on the considered cell lines, showing GI50 values in the low micromolar range.
The results obtained so far indicate the benzothiopyranoindole derivatives are potential dual topoisomerase I and II inhibitors, while the pyridothiopyranoindole system seems to be suitable for obtaining efficacious topoisomerase I poisons.
Unfortunately, intrinsic and acquired mechanism of resistance take place their ability makes them the most important factor responsible for the rise of resistance phenomena against Topo I and Topo II inhibitors, since these enzymes break the cleavage complex between topoisomerases and their inhibitors. Tyrosyl-DNA phosphodiesterases (TDP1 and TDP2), the most recently discovered DNA repair enzymes, have gain attention from researchers in developing new anticancer compounds. Their physiological role is to liberate DNA ends from the covalently stalled topoisomerase by cleaving the covalent phosphotyrosyl bond linking the topoisomerase to DNA, a process that is tightly regulated by post-translational protein modifications. Eukaryotes possess two distinct TDPs as defined by their enzymatic activities in vitro. These are a metal independent TDP1, which primarily acts on DNA breaks with 3′- phosphotyrosyl termini, and a metal-dependent TDP2, which acts on DNA breaks with 5′- phosphotyrosyl termini.
So, Tdp1 has been regarded as a potential co-target of Top1 for anticancer therapy, in that it seemingly counteracts the effects of Top1 inhibitors, such as camptothecin and its clinically used derivatives. In this view, Tdp1 inhibitors have the potential to enhance the anticancer activity of Top1 inhibitors, by reducing the repair of Top1-DNA lesions.
Within a project aimed to identify new potential Tdp1 inhibitors, in collaboration with the group of Professor Pommier of NIH (Bethesda) an in vitro screening on an in-house library of structurally heterogeneous chemical compounds was performed. Three of these showed weak inhibitory activity on Tdp1, so representing lead compounds to be further improved by a lead optimization process.
These compounds were used as a starting point for a de novo design strategy, by means of computational studies conducted in collaboration with the research group of Professor Novellino (University of Naples).
Thus, guided by molecular modeling studies on TDP24, a small library of benzothiopyranoindole derivatives, substituted with hydroxylic groups at different positions, has been designed, along with a set of indolglyoxylethylester, indolglyoxylamide, indole featuring an ester moiety and an amide moiety at position 3 derivatives substituted with alcoholic and amine chains of different lengths at specific positions.
Biological results on both Tdp1 and Tdp2 enzymes from our collaborators at NIH showed that benzothiopyranoindole derivatives possess activity in the micromolar range against Tdp1 while they are inactive towards Tdp2. The other compounds proved to be inactive against both the enzymes. These results suggest a selectivity of benzothiopyranoindole towards Tdp1 and further studies should be conducted in order to increase potency of this promising scaffold.