Synthesis of constrained peptidomimetics for therapeutic, diagnostic and theranostic applications

This thesis work deals, principally, with the development of different chemical protocols ranging from environmental sustainability peptide synthesis to asymmetric synthesis of modified tryptophans to a series of straightforward procedures for constraining peptide backbones without the need for a pre-formed scaffold. Much efforts have been dedicated to the structural analysis in a biomimetic environment, fundamental for predicting the in vivo conformation of compounds, as well as for giving a rationale to the experimentally determined bioactivity. The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, titration experiments, molecular dynamics, etc.), FT-IR and ECD spectroscopy. As a practical application, 3D rigid scaffolds have been employed for the synthesis of biological active compounds based on peptidomimetic and retro-mimetic structures. These mimics have been investigated for their potential as antiflammatory agents and actually the results obtained are very promising. Moreover, the synthesis of Amo ring permitted the development of an alternative high effective synthetic pathway for obtaining Linezolid antibiotic. The final section is, instead, dedicated to the construction of a new biosensor based on zeolite L SAMs functionalized with the integrin ligand c[RGDfK], that has showed high efficiency for the selective detection of tumor cells. Such kind of sensor could, in fact, enable the convenient, non-invasive detection and diagnosis of cancer in early stages, from a few drops of a patient's blood or other biological fluids. In conclusion, the researches described herein demonstrate that the peptidomimetic approach to 3D definite structures, allows unambiguous investigation of the structure-activity relationships, giving an access to a wide range bioactive compounds of pharmaceutical interest to use not only as potential drugs but also for diagnostic and theranostic applications.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

Synthesis of Modified Amino Acids and Insertion in Peptides and Mimetics. Structural Aspects and Impact on Biological Activity.

I studied the effects exerted by the modifications on structures and biological activities of the compounds so obtained. I prepared peptide analogues containing unusual amino acids such as halogenated, alkylated (S)- or (R)-tryptophans, useful for the synthesis of mimetics of the endogenous opioid peptide endomorphin-1, or 2-oxo-1,3-oxazolidine-4-carboxylic acids, utilized as pseudo-prolines having a clear all-trans configuration of the preceding peptide bond. The latter gave access to a series of constrained peptidomimetics with potential interest in medicinal chemistry and in the field of the foldamers. In particular, I have dedicated much efforts to the preparation of cyclopentapeptides containing D-configured, alfa-, or beta-aminoacids, and also of cyclotetrapeptides including the retro-inverso modification. The conformational analyses confirmed that these cyclic compounds can be utilized as rigid scaffolds mimicking gamma- or beta-turns, allowing to generate new molecular and 3D diversity. Much work has been dedicated to the structural analysis in solution and in the receptor-bound state, fundamental for giving a rationale to the experimentally determined bioactivity, as well as for predicting the activity of virtual compounds (in silico pre-screen). The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, molecular dynamics, etc.). A special section is dedicated to the prediction of plausible poses of the ligands when bound to the receptors by Molecular Docking. This computational method proved to be a powerful tool for the investigation of ligand-receptor interactions, and for the design of selective agonists and antagonists. Another practical use of cyclic peptidomimetics was the synthesis and biological evaluation of cyclic analogues of endomorphin-1 lacking in a protonable amino group. The studies revealed that a inverse type II beta-turn on D-Trp-Phe constituted the bioactive conformation.

Synthesis and surface modification of silver and gold nanoparticles. Nanomedicine applications against Glioblastoma Multiforme.

In the last decades noble metal nanoparticles (NPs) arose as one of the most powerful tools for applications in nanomedicine field and cancer treatment. Glioblastoma multiforme (GBM), in particular, is one of the most aggressive malignant brain tumors that nowadays still presents a dramatic scenario concerning median survival. Gold nanorods (GNRs) and silver nanoparticles (AgNPs) could find applications such as diagnostic imaging, hyperthermia and glioblastoma therapy. During these three years, both GNRs and AgNPs were synthesized with the “salt reduction” method and, through a novel double phase transfer process, using specifically designed thiol-based ligands, lipophilic GNRs and AgNPs were obtained and separately entrapped into biocompatible and biodegradable PEG-based polymeric nanoparticles (PNPs) suitable for drug delivery within the body. Moreover, a synergistic effect of AgNPs with the Alisertib drug, were investigated thanks to the simultaneous entrapment of these two moieties into PNPs. In addition, Chlorotoxin (Cltx), a peptide that specifically recognize brain cancer cells, was conjugated onto the external surface of PNPs. The so-obtained novel nanosystems were evaluated for in vitro and in vivo applications against glioblastoma multiforme. In particular, for GNRs-PNPs, their safety, their suitability as optoacoustic contrast agents, their selective laser-induced cells death and finally, a high tumor retention were all demonstrated. Concerning AgNPs-PNPs, promising tumor toxicity and a strong synergistic effect with Alisertib was observed (IC50 10 nM), as well as good in vivo biodistribution, high tumor uptake and significative tumor reduction in tumor bearing mice. Finally, the two nanostructures were linked together, through an organic framework, exploiting the click chemistry azido-alkyne Huisgen cycloaddition, between two ligands previously attached to the NPs surface; this multifunctional complex nanosystem was successfully entrapped into PNPs with nanoparticles’ properties maintenance, obtaining in this way a powerful and promising tool for cancer fight and defeat.