Novel synthetic pathways for the preparation of ProTides as potential therapeutic agents

The phosphoroamidate (ProTide) approach is a prodrug technology aimed to circumvent metabolic bottlenecks in the activation of nucleoside-based drugs and optimise their intracellular delivery. The tremendous importance of the ProTide approach is highlighted by the approval of Sofosbuvir (Sovaldi®, HCV infections) and tenofovir alafenamide fumarate (TAF, Vemlidy®, HIV and HBV infections). A great deal of success is also demonstrated by many other compounds adopting this technology either in clinical trials or preclinical evaluations as antiviral and anticancer agents. Given the great impact of phosphor(n)oamidate nucleoside prodrugs in the antiviral arena and beyond, the application of this technology has grown dramatically. Several procedures are present in the literature for the preparation of phosphoroamidate prodrugs of nucleosides. However, an efficient and inexpensive diastereoselective synthesis to prepare ProTides as single diastereoisomers is missing. Additionally, the phosphonoamidate cognate class, one of the most significant groups of antiviral drugs, presents many synthetic challenges. Recent literature reported the synthesis of novel acyclic nucleoside backbones including the phosphonate derivatives bearing a double bond in the aliphatic chain. However, the methodologies described for the preparation of ProTides on alkenyl acyclic nucleosides are scarce and inefficient. Beside phosphoroamidates and phosphonoamidates, many difficulties can also be encountered in the preparation of modified unnatural nucleosides and related prodrugs. One of them is the ProTide of 2’-deoxy-O6-methylguanosine to be tested for mitochondrial DNA depletion syndrome. In this context, the research discussed in this thesis is focused on addressing the synthetic problems related to unnatural nucleosides and their ProTides. This thesis aims to explore novel methodologies for the preparation of both phosphoroamidate and phosphonoamidate prodrugs of biologically relevant nucleosides in order to give easy access to novel ProTides to be evaluated for their potential therapeutic act

Preparation of pyrimidine alkenyl acyclic nucleoside phosphonoamidates

This synthetic protocol describes two strategies for the preparation of pyrimidine alkenyl acyclic nucleoside phosphonoamidates (ANPs), including linear and trisubstituted alkenyl derivatives. For the first procedure, a bis‐trimethylsilyl ester of the parent alkenyl ANPs is the key intermediate that reacts with the desired amino acid ester and aryl alcohol. For the second procedure, an allyl phosphonoamidate bearing the ProTide promoieties is the key synthon employed as olefin partner for a cross‐metathesis reaction with an alkylated nucleobase

Expedient synthesis and biological evaluation of alkenyl acyclic nucleoside phosphonate prodrugs

The importance of phosphonoamidate prodrugs (ProTides) of acyclic nucleoside phosphonate (ANPs) is highlighted by the approval of Tenofovir Alafenamide Fumarate for the treatment of HIV and HBV infections. In the present paper we are reporting an expedient, one-pot, two-steps synthesis of allyl phosphonoamidates and diamidates that offers a time saving strategy when compared to literature methods. The use of these substrates in the cross metathesis reactions with alkenyl functionalised thymine and uracil nucleobases is reported. ANPs prodrugs synthesized via this methodology were evaluated for their antiviral activities against DNA and RNA viruses. It is anticipated that the use of 5,6,7,8-tetrahydro-1-napthyl as aryloxy moiety is capable to confer antiviral activity among a series of otherwise inactive uracil ProTides

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA

Computational studies towards the identification of novel rhodopsin-binding compounds as chemical chaperones for misfolded opsins

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber’s congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber’s congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases

Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials

Objectives Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. Methods Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. Results The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. Conclusions Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria