Phd in PHARMACEUTICAL SCIENCES

Thesis title: Syntheses of natural products: from bench to preclinical studies

This PhD thesis is focused on the role of natural products in drug discovery and it is divided into two main parts (part A and B). Part A investigates the role of natural compounds in inhibiting Hedgehog (Hh) and Notch signaling pathways which overactivation is responsible for the onset of several cancers. In particular, an in house library of more one thousand natural products has been screened and the chalcone Butein and the isoflavone Glabrescione B (GlaB) emerged as hit compounds for the modulation of Notch and Hedgehog pathway, respectively. Based on SAR studies of compound 8, the most potent Notch inhibitor of a first series of chalcone derivatives previously synthesized in our laboratory, a second generation of chalcones and chalcone mimetics was selected, synthesized, and tested. Chalcone 1 and chalcone mimetic 18 exhibited a considerable cell growth reduction combined with a strong inhibition of Notch1 intracellular domain, mostly comparable to the reference compound 8, suggesting that further investigations are needed to develop new chalcone derivatives as Notch blocking agents. On the other hand, Glabrescione B (GlaB) resulted as the most promising downstream inhibitor of Hedgehog signaling pathway due to its strong interaction with Gli1 factors. In this work, our most promising pharmacophores targeting Smo and Gli1 were combined in a single and individual isoflavone and a new class of multitarget Hh inhibitors was designed and synthesized. Compound 22 resulted the most potent isoflavone able to inhibits the Hh pathway at both upstream (Smo receptor) and downstream level (Gli factors), suppressing medulloblastoma (MB) growth in vitro and in vivo through antagonism of Smo and Gli1. This novel mechanism of action in Hh inhibition encourages the use of a multitargeting approach for the treatment of Hh-driven tumors. The hit compound GlaB also emerged as a good candidate for pre-clinical investigations in the treatment of Hh-driven cancers (MB and BCC). To overcome its poor solubility, GlaB was formulated with self-assembling amphiphilic polymers forming micelles, namely mPEG5kDa-cholane and in vivo studies in both allograft and orthotopic models of Hh-dependent MB were conducted. The biodistribution of GlaB/mPEG5kDa-cholane in brain and cerebellum was assessed by High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS). HPLC analysis on brain and cerebellum, revealed that the drug loaded in mPEG5kDa-cholane based micelles upon intravenous administration crosses the BBB, reducing in situ MB growth and suggesting this formulation as an interesting candidate for clinical studies for the treatment of Hh-dependent cancers, such Hh-MB. Part B focuses on the development of new synthetical strategies for the replication of the architectural complexity of polyketides, a family of natural compounds endowed with biological activity. During the years, numerous synthetic approaches have been advanced and among all organic reactions, aldol condensation emerged as an excellent method for the stereoselective construction of the polyketide backbone. Very interesting is the vinylogous Mukaiyama aldol reaction (VMAR), an enantioselective and catalytic aldol additions of silyl enol ethers with carbonyl compounds in the presence of Lewis acids. The final product of VMA reaction is a δ-alkoxy-α,β-unsaturated aldehyde in which the double bond can undergo sequential regio- and stereoselective reductions, providing an efficient method to synthesize reduced polypropionate, a common feature of natural products with polyketide structures. The methods to realize the short step synthesis of the reduced polypropionates has always represented a powerful tool to obtain bioactive compounds. In this thesis is presented a concise and stereochemically flexible synthesis of a reduced polypropionate chain.