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In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this

My PhD research project has been finalized to the discovery of potential RAD51-BRCA2 protein-protein interaction (PPI) disruptors, able to trigger synthetic lethality (SL) in pancreatic cancer in combination with PARP inhibitor Olaparib. SL has been validated clinically, as anticancer therapeutic strategy, through the efficacy of Olaparib in BRCA2-defective cancers. In this context, we proposed to trigger a fully small-molecule-induced SL, combining RAD51-BRCA2 PPI disruptor with Olaparib to target pancreatic cancer. RAD51-BRCA2 PPI is essential in homologous recombination (HR) for DNA repair and it is mediated by two critical “hotspots” on RAD51 surface, Zone I and Zone II, suitable sites to design small molecule disruptors. This thesis reports the application of two distinct hit identification strategies to identify potential RAD51-BRCA2 PPI disruptors. In the first part of this project, an approach of structure-based design was applied, which exploited Virtual Screening (VS) campaign as hit identification strategy, followed by chemical modifications and SAR studies. Two VS, targeting separately Zone I and Zone II, led to the discovery of two classes of potential RAD51-BRCA2 PPI disruptors. In particular, VS targeting Zone II identified the dihydroquinolone pyrazoline 19 as hit compound. The optimization of a general synthetic strategy gave access to a series of analogs for SAR investigation, leading to compound 47 with the desired biological profile. Indeed, 47 inhibited RAD51-BRCA2 PPI, reduced cell HR and triggered cell death in combination with Olaparib in BxPC-3 cells, fully reproducing the paradigm of SL. The second part of this thesis was devoted to a second hit identification strategy. During the six-month placement at HIPS in Prof. A. K. H. Hirsch’s group, I applied a technique of target directed dynamic combinatorial chemistry (tdDCC) on RAD51. This approach allowed the identification of N-acylhydrazone-based compounds, which proved to inhibit RAD51-BRCA

This PhD project was carried out within the frame of a Marie Curie Network with the acronym ‘ClickGene’, which focuses on the development of next-generation gene silencing therapeutics, such as the artificial metallo-nucleases (AMNs). So far, the molecular basis of the drugs mechanism has focused primarily on DNA damage, but recently the novel antitumoral strategies highlighted the cell membranes as a relevant site of the drug’s multitarget reactivity towards the unsaturated lipids. The PhD thesis is devoted to the study of the oxidation mechanisms of both lipids and nucleic acids induced by free radicals in the presence of AMN. The work first aimed to investigate the lipid reactivity in the presence of a metallo-drug. To simulate the cell membrane, a biomimetic model of liposomes was designed by different mono- and poly-unsaturated fatty acid moieties in order to examine the reactivity in the presence of the novel synthesized AMN and a reducing agent. A variety of conditions was tested giving an interesting mechanistic picture of the membrane-drug interaction. Connected with the fatty acid transformations, the novel synthesis and the full analytical characterization of the six mono-trans isomers of docosahexaenoic acid (DHA) was performed. Two different synthetic approaches were combined to obtain a full identification of the six mono-trans isomers and the analysis was based on GC and NMR for building-up a molecular reference library. In the second part of the project, the work focused on AMN ability for precise cleavage of DNA. In these experiments, the damage profiles were studied using major groove recognition elements and spin-trapping scavengers of ROS. The DNA damage fragments were purified and enzymatically digested to single nucleosides. Finally, the analytical protocol established by our group was applied to identify the drug effect regarding the formation of oxidative lesions, in double strand DNA fragments.

The present work includes distinct research projects having as common platform the design and synthesis of biologically active small molecules, useful as tools both for drug discovery and chemical biology studies. The main project (Part I) focused on the identification of LDH-A inhibitors as hit candidates in the discovery of new anticancer agents. My work aimed to explore the chemical space around galloflavin (GF) by synthesizing a small library of analogs to perform structure-activity relationships (SAR) studies. GF, LDH-A inhibitor previously identified and synthesized by our group, was not investigated in terms of SAR because of its poor chemical tractability. In the present work, natural urolithin M6 (UM6) has been selected as simplified analog of GF. It is endowed with improved physicochemical properties and showed to reproduce GF’s biological behaviour. An efficient synthetic strategy has been developed to obtain UM6, which synthesis was not previously reported. This route gave an easy access to a series of structural analogs for SAR investigation. The second project (Part II), carried out at Oxford University under the supervision of Professor Angela Russell, aimed to use a chemical biology approach to develop a proneurogenic small molecule recently identified by the group (OX02672), into a fluorescent probe to assess its molecular interactions and localization in neural stem cells (NSCs). My work focused on the design and synthesis of alkyne-tagged derivatives of OX02672 for subsequent development into probes by “click” conjugation with azide-containing fluorophores. The third part includes a side-project (Part III), carried out in collaboration with group of Professor Maria Laura Bolognesi. It focused on the design and synthesis of multi-target-directed ligands (MTDLs) useful as potential hit candidates in the search for new drugs against Alzheimer’s disease (AD). Tacrine-resveratrol hybrid compounds have been designed and synthesized by using a fusing str

The present work aimed to synthesizing new biologically active small molecules as innovative antitumor lead candidates and to the total synthesis of a natural compound with selectivity towards cancer hypoxia. In this context, a first project involved the design and synthesis of N-acylhydrazone based inhibitors of lactate dehydrogenase A (LDH-A). The structures of the new molecules where designed by means of virtual screening and synthesized to obtain a library of analogs which were evaluated on the enzyme. Active compounds were also screened on cells of non-Hodgkins lymphoma and one of them proved to be a promising inhibitor, suggesting that the N-acylhydrazone as suitable scaffolds for LDH-A inhibitors. The second project aimed to the synthesis of Galloflavin (GF) analogs and to the study of the compound’s SAR. GF is an LDH-A inhibitor which was previously identified and synthesized by our group. Its poor solubility and stability prevented us from studying its SAR maintaining the core structure. Therefore, the synthesis of three potential classes of structural analogs was devised and carried out. One compound was found to reproduce GF’s behaviour on the enzyme and in cell, therefore being a good starting point for the study. A small library of analogs was synthesized and biological tests are ongoing to acquire in-depth knowledge about the key pharmacophores of this interesting inhibitor. The third project was carried out at Aarhus University in the group of Prof. Thomas Poulsen. The work focused on the total synthesis of Rakicidin A, a macrolide of natural origin which was identified and isolated from soil samples and is known for its interesting properties in selectively inducing cell death in hypoxic environments and being also active on cancer stem cells. The total synthesis involved several steps including key enantioselective reactions to build the 5 stereocenters on the molecule.