Phd in LIFE SCIENCE

Thesis title: Design, synthesis and biological evaluation of new heterocycles endowed with antitumoral activity

My Ph.D. thesis is focused on the design and synthesis of new heterocycle-based small molecules endowed with antitumoral activity. Heparanase inhibitors The first part of my thesis is focused on Heparanase (Hpse), the sole mammalian enzyme able to cleave glycosaminoglycan heparan sulfate side chains of heparan sulfate proteoglycans, with retention of the anomeric configuration. Proteoglycans participate in several biological activities by interacting with various enzymes or receptors. By editing their structures, Hpse is consequently involved in a multitude of vital cellular pathways, but its aberrant activity is associated with tumor development and negative prognosis. Indeed, it contributes to all known hallmarks of cancer, thus making it an attractive pharmacological target. However, although the interest in this enzyme is enormously grown in the past years and various attempts have been made to develop new Hpse inhibitors, to date no Hpse small-molecule-based inhibitors have ever entered clinical trials. Hence, I designed and synthesized two libraries of benzimidazole- and benzoxazole-based molecules, characterized by both symmetrical and asymmetrical structures. These molecules were conceived by merging the pharmacophoric moieties of known Hpse small-molecule-based in vitro inhibitors and by functionalizing the terminal 5-acetyl groups with amino acids or amino alkyl boronic groups. Fluorine-hydrogen atom substitutions were also evaluated in the central aniline rings, and both ureidic and thioureidic central groups were synthesized. The majority of newly synthesized compounds were found active in in vitro Hpse inhibition assays with micromolar concentrations, with symmetric compounds showing the better potencies. Among them, a glycine conjugated thiourea benzoxazole exhibited the best inhibitory activity found, with an IC50 of 80 nM. By comparing the activity of this compound to that of the polysaccharide roneparstat, one of the most potent Hpse inhibitors reported, is evident that, although the newly synthesized thioureidic benzoxazole is 16 times less potent than roneparstat if the activity is expressed in molar concentration, the amount potentially necessary for its clinical use could be about the half of that of roneparstat due to their huge difference in molecular weight, highlighting the great versatility and suitability of small molecules in the development of new Hpse inhibitors. Through computational studies, new compounds were demonstrated to act as orthosteric competitive inhibitors, lying within the Hpse substrate binding cleft and occupying the catalytic site. Heparin binding domain 2 (HBD-2) appears as the major anchoring point for the amino acids asymmetric benzazoles, while the symmetric compounds place the urea or thiourea group close to the catalytic residue E225 and contact both the Hpse HBD-1 and HBD-2, thus undertaking a higher number of polar interactions. Furthermore, some compounds proved also to inhibit cell proliferation and invasive potential on HT1080, U87MG and U2OS tumor cell lines and the transcription of genes encoding for proangiogenic factors, such as MMP-9, VEGF, and FGFs in tumor cells. More in detail, in U87MG cell line, the most potent Hpse inhibitor found was also demonstrated to induce the autophagosomes accumulation and the arrest in the autophagic flux. Consistently, the treatment with the newly synthesized inhibitor might compromise the pro-autophagy function of the intracellular Hpse, considering the ability of the inhibitor to easily cross the cellular plasma membrane. Protein kinase inhibitors The second part of my thesis regards the design and synthesis of new series of small-molecule protein kinase inhibitors. Protein kinases catalyze the transfer of γ-phosphates from ATP to an amino acid -OH group of a substrate protein, and according to the amino acid nature, they could be classified as serine/threonine or tyrosine protein kinases. Protein kinases are intimately involved in many physiological activities, but their deregulation has widely recognized to be a relevant mechanism by which cancer cells evade normal constraints and adopt tumorigenic properties. Hence, the discovery of protein kinases inhibitors has become one of the most intensively pursued classes of recent drugs, capturing funding for billions of dollars worldwide. In this field, pyrimidines have received much interest due to their intrinsic biological potential and many pyrimidine-based derivatives possess promising anticancer activity thanks to the inhibition of the protein kinases catalytic site. In this context, I designed and synthesized two classes of compounds, 2,4,6-tris-amino pyrimidines and 2-anilino pyrimidines. A 2,4,6-tris-amino pyrimidine derivative was previously identified as an inhibitor of replication, a negative regulator of cell cycle progression and an inducer of apoptosis in U87MG, MDA-MB231, and HT-29 cell histotypes. In order to verify if the mechanism of action of this compound is due to a protein kinase inhibition and to investigate the SAR, new structurally related derivatives were conceived by carrying out substitutions in position 6 of the pyrimidine core and/or on the 2-aniline ring and by derivatizing the 2-NH- group of pyrimidine with a p-fluorobenzyl ring. The new N-benzylated compounds proved to be generally endowed with higher potencies than the hit compound, by causing a more pronounced decrease in cell viability. The best acting compound reported EC50 values ranging from 10 to 26 μM at 24 h of treatment and from 5 to 8 μM at 48 h of treatment, 13-fold higher than the starting compound, against all the tested cell lines. Among this series, considering the substitution in meta position of aniline ring, propanediamine derivatives activities decreased in the following order: Cl > F > NO2 ≈ OCH3, indicating the importance of an electron withdrawing group. These compounds were also tested to verify their protein kinases inhibition activities by HTRF assay, and they were found to hinder EGFR, wt and mutated isoforms, with nanomolar activities. The 2-anilino pyrimidines, instead, were tailor-designed to mostly interact with Aurora kinases A (AURKA). More in detail, I investigated and expanded this scaffold by studying three sub-classes of compounds: (1) 4-(pyrimidin-2-ylamino)benzoic acids, to be orthosteric competitive inhibitors of AURKA; (2) protein-protein interaction inhibitors of AURKA/N-Myc interaction, characterized by an AURKA orthosteric competitive core and an electrophilic warhead to covalently bind to the AURKA Cys290 in order to induce an AURKA conformational change to prevent N-Myc binding; and (3) dual kinases inhibitors, of AURKA and HDAC, merging the structures of previously AURKA inhibitors identified and HDAC inhibitor quisinostat. All the newly designed compounds were synthesized and evaluated to inhibit AURKA in in vitro ADP-Glo assay. The majority of compounds were found active with nanomolar concentration and most of them were also co-crystallized in the active site of the enzyme. Considering the second group of compounds, fumaric acid derivative inhibited AURKA and also other kinases, namely c-KIT, PDGFR wt and mutated isoforms and VEGFR2, in in vitro HTRF assay, emerging as an innovative chemical scaffold for the development of a new pluri-kinase inhibitor, particularly useful for those disease in which PDGFR and VEGRF2 cooperate to overcome Bevacizumab efficacy. Anyway, other biological studies to evaluate the AURKA/N-Myc interaction inhibition and the covalent-binding capabilities are under investigation. Regarding the last group of protein inhibitors, while the benzoic acid derivative inhibited AURKA in concentrations lower than every other AURKA inhibitor to date described, the hydroxamic acid counterparts showed lower AURKA inhibition activities but, at the same time, micromolar HDAC inhibition values, acting as potential dual kinases inhibitors. To conclude, in this Ph.D. thesis, I designed and synthesized two libraries of compounds. Benzimidazole- and benzoxazole-based molecules were found active against Hpse, in in vitro assays, with symmetric derivatives showing the best potencies. Moreover, they also inhibited cell proliferation, invasive potential, and the transcription of several pro-tumorigenic genes in the tested cancer lines, and act as a balance between autophagy and apoptosis. Considering the pyrimidine-based compounds, the first group of newly designed and synthesized N-benzylated 2,4,6-trisubstitued pyrimidines functions as antitumoral compounds on various tested cell lines and exerted pronounced EGFR wt and mutated isoforms inhibition activities. The 2-anilino pyrimidine, instead, act as AURKA orthosteric inhibitors, and their chemical tailored-modifications allow to selective modulate their activity profile, spacing from the pluri-kinase inhibitor fumaric acid to the dual AURKA/HDAC inhibitors. The most promising compounds lend themselves to be optimized to further hit-to-lead development processes.