Pagano, Rita (2024) Synthesis and characterization of new drugs inspired by natural products. [Tesi di dottorato]

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Abstract

Natural products (NPs) have always been a source of lead compounds that promote pharmacological advances to treat different health problems, mainly related to cancer, infectious diseases, inflammation, neurodegeneration and cardiovascular disorders. Among NPs, plant polyphenols are gaining increasing recognition thanks to their potent antioxidant properties, and their therapeutic advantages in the modulation of cell signaling, anti-inflammatory effects and neuroprotection. Despite their widespread distribution and different benefits, the poor bioavailability of flavonoids may significantly impact their therapeutic effects. For these reasons, the research is active in identifying more common or easily synthetic flavonoids with enhanced absorption, better therapeutic efficacy and fewer side effects. During my PhD I focused my attention on two natural products, silibinin and curcumin, both of plant origin which simultaneously suffer from very low solubility in water and poor bioavailability. Silibinin is the main component of the silymarin extracted from milk thistle seeds and consists of an approximately equimolar mixture of two diastereoisomers: silybin A (Sil A) and silybin B (Sil B). Silibinin, known above all for its hepatoprotective activity, has a wide range of biological and pharmacological activities, but it’s limited by its low solubility in water and bioavailability. In many cases the optical pure aspect of this two diastereoisomers has been largely neglected, but some studies have demonstrated that stereochemistry matters when describing their biological effects. A recent study, indeed, investigated the silybins ability to inhibit Aβ amyloid growth and toxicity, and demonstrated that only Sil B was able to significantly inhibit Aβ aggregation in aqueous solutions and counteract Aβ proteotoxicity in worms (Caenorhabditis elegans) expressing human Aβ. Conversely, in the case of human 20S proteasome (h20S) Sil A demonstrated a higher affinity and more efficient activation than Sil B. Curcumin is the bioactive compound of Curcuma longa. Its pharmacological action has been mainly attributed to its antioxidant and anticancer activities, ability to chelate bio-metals and to inhibit the aggregation of the Aβ peptide. Despite such potential, curcumin suffer of low bioavailability and poor chemical stability that limit its pharmacological applications. Several strategies were developed to overcome these disadvantages, such as nanoparticle and formulations in liposomal complex. One of the most common strategies is the design and synthesis of new curcumin mimics with better therapeutic properties and bioavailability. In this frame, during my PhD I was involved in the synthesis, characterization and evaluation of different biological activities of new silybin derivatives and curcumin mimics. Silybin derivatives To increase water solubility of silybins, new prodrugs were designed. A mini-library of 9′′-silybins conjugated with 3′-ribonucleotide units through phosphodiester junctions was synthesized with an efficient regioselective solid-phase synthetic approach. The new prodrugs showed greater solubility in water (3.7 mM and 6 mM for uridine and adenosine conjugates, respectively) compared to silybins (1 µM). Investigations performed to validate the effective timed-release of new prodrugs have revealed that uridine-silybin derivatives were quickly cleaved by RNase A, releasing the active silybin drugs even at low RNase concentrations. MTT assays confirmed the lack of toxicity of the new compounds on neuronal cancer cells. A recent study reported the synthesis and antioxidant activity of bi-flavonoids based on silibinin. The new dimers, linked through a phosphodiester bond in position 3-3, 3-9" and 9"-9", have displayed high ability to scavenge ROS, comparable to the value reported for known potent antioxidants such as quercetin. In this regard, during my PhD I investigate the structure–activity relationships of dimer 9"-9", which turned out to be the best one. Starting from diastereoisomerically pure silybin monomers (Sil A and Sil B), employing orthogonal protection of various hydroxyl groups and the phosphoramidite chemistry, novel 9"-9" dimers of silybin A and silybin B (7aa, 7ab, and 7bb) were successfully synthesized. They showed a higher ability to scavenge reactive oxygen species (ROS), specifically the hydroxyl radical (HO•), comparable to quercetin. Investigations on the anticancer activity of new dimers, reveal that both monomers and dimers display selective anti-proliferative activity against leukemia cells at the concentrations employed, and low activity on normal cells. However, the cytotoxic mechanisms is not apoptosis for everyone, underlighting the pivotal role of stereochemistry. Considering the inhibitory effects on various cancer cells of silibinin and its oxidation product 2,3-dehydrosilybin (DHS), chemical modification studies could enhance their bioavailability and antiproliferative activity against prostate cancer cell lines. For this reason, it was developed a regioselective synthesis of new 7-O-alkyl derivatives aims to combine the pharmacological properties of silybins and DHS with tyrosol-based metabolites, potentially yielding compounds with improved antioxidant and anticancer activities. While in the ORAC assay there was no significant variation in antioxidant activity, in the DPPH assays, the new derivatives exhibited pronounced activity due to the role of tyrosolic counterpart. From the study on PC-3 prostate cancer cells emerged that the compounds DHS-HTYR, DHSA-HTYR and DHSB-HTYR inhibit cell proliferation and induce cell death in PC-3 cells. Curcumin mimics With the aim of synthesizing curcumin mimics with broad molecular diversity, it has been carried out a rapid solid-phase synthetic strategy to obtain tyrosol-based phosphodiester mimics which retain the two aromatic rings with distinct hydroxyl substituents and a distance between them comparable to that of curcumin. The phosphodiester linker was chosen with the aim of increasing the water solubility and the stability of the new mimics. From the study emerged that all new mimics exhibited high stability in simulated intestinal fluid, simulated gastric fluid, alkaline phosphatase, and serum buffers. The phosphodiester dimers potent growth inhibition and cell death efficacy in PC-3 prostate cancer cells for the compound tyrosol with a homovanillil substitution, demonstrating a substantial effectiveness in cell growth inhibition and in inducing significant cell death. To expand this first library, new tyrosol-based ethyl phosphonates curcumin mimics were synthesized. The new mimics retain the phenolic moieties based on tyrosol, homovanillil and hydroxytyrosol alcohols, in addition to the 5-methoxy tryptophol. The linker was replaced with an uncharged ethyl phosphonate with a length of seven atoms. The mimics EP2 and EP4 resulted the most interesting. EP2 displayed strong antioxidant activity and potent inhibitory effect on ferroptosis, representing a potential candidate for neurodegenerative disorders. Conversely, EP4 is a very potent anticancer agent against different human cancer cell lines (HeLa, A375, WM266, MDA-MB-231) and no cytotoxic on normal cell (HDF). Mechanistic investigation suggested that EP4-induced cell death occurs primarily by apoptosis.

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