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IN SEARCH FOR SUGAR, BASE OR PHOSPHATE MODIFIED NUCLEOSIDES ACTIVE AGAINST RNA VIRUSES

Nucleoside and nucleotide analogues obtained from the chemical modification of naturally occurring building blocks may exhibit interesting biological properties, such as potent antitumor, antiviral, and immunosuppressive activities. In particular, nucleoside analogues and their prodrugs constitute a well-established and essential class of antiviral drugs used in the clinic for the treatment of certain infectious diseases caused by DNA and RNA viruses such as herpes simplex virus (HSV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV). Once activated in the cell to their active phosphorylated forms, nucleosides can interfere with viral replication by inhibiting important enzymes such as viral polymerases or undergoing incorporation into a growing nucleic acid chain leading to elongation termination. Owing to the highly conserved binding site of polymerases, nucleos(t)ide analogues may display potential broad-spectrum antiviral activity. The development of broad-spectrum antivirals is crucial for facing newly emerging or re-emerging viral infectious diseases. Increasing the structural diversity by means of chemical modifications is a powerful tool to enrich the currently available library of antiviral nucleos(t)ide analogues and plays a key role in the disclosure of promising lead candidates.

The main focus of this thesis lies on identifying novel potentially active and low cytotoxic antiviral nucleoside agents. To this end, we selected specific structural variations that led to a variety of modified nucleosides including 4’-substituted analogues of the anticancer drug gemcitabine, 6- and 6,7-substituted derivatives of the antibiotic tubercidin (7-deazaadenosine), 7-substituted 4-aza-7,9-dideazaadenosine C-nucleoside analogues, as well as l/d-4’-thionucleoside phosphonates.

The synthesis of 4’-substituted gemcitabine analogues along with that of their phosphoramidate prodrugs is described in Chapter 2. In this study, 4’,5’-unsaturated 2’,2’-difluoro-2’-deoxyuridine was used as key synthon for the stereoselective introduction of methoxy and fluorine groups at the 4’-position. The antiviral activity of the resulting derivatives against various DNA viruses, such as varicella zoster virus (VZV) and human cytomegalovirus (HCMV), as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was evaluated as well as their cytotoxicity. The trifluorinated analogue 4’-fluorogemcitabine exhibited potent antiviral activity against VZV (EC50 = 0.042 mM) albeit with significant cytotoxicity (EC50 = 0.11 mM), while its phosphoramidate prodrug showed an improved selectivity index (SI50 = 36). Unfortunately, no compounds displayed promising activity against SARS-CoV-2.

In Chapter 3, the chemical synthesis of 6-substituted and 6,7-disubstituted analogues of 7-deazaadenosine is described. The introduction of different substituents at the 6-position was carried out via metal-catalyzed coupling reactions in the presence of either Fe/Cu or palladium-based catalysts. The resulting two series of base-modified 7-deazaadenosine derivatives were screened for their antiviral activity against a panel of RNA viruses and their cytotoxicity was also determined. Among 6-substituted derivatives, 6-isopropyl, 6-(4-isopropyl)phenyl, and 6-(4-fluorophenyl)ethynyl analogues exhibited highly selective nanomolar activity against human norovirus (HuNoV) in a replicon system. Notably, the 6-cyclopropylethynyl analogue displayed broad-spectrum antiviral activity against different RNA viruses, such as yellow fever virus (YFV), Middle East respiratory syndrome coronavirus (MERS-CoV), measles virus, and tacaribe virus. Furthermore, the 6-ethyl and 6-ethyl-7-fluoro-7-deazaadenosine analogues displayed potent antiviral activity and high selectivity against influenza virus. Overall, the introduction of small modifications at the nucleobase of 7-deazaadenosine is an effective strategy to generate potent inhibitors of RNA viruses with broad-spectrum activity.

In Chapter 4, a series of 7-substituted analogues of the C-nucleoside 4-aza-7,9-dideazaadenosine were synthesized starting from 7-iodo-4-aza-7,9-dideazaadenosine via palladium-catalyzed coupling reactions. Upon evaluation of their antiviral activity and cytotoxicity, the 7-vinyl and 7-(4-methylstyryl) 4-aza-7,9-dideazaadenosine analogues emerged as promising compounds, showing submicromolar activity against SARS-CoV-2. In particular, 7-(4-methylstyryl)-4-aza-7,9-dideazaadenosine exhibited also good selectivity (SI50 = 150) in Vero 76 cells. Further optimization of the antiviral efficacy of these compounds and the investigation of their mode of action will be the subject of future studies.

In the final part of this thesis (Chapter 5), the first synthesis of 4’-thionucleoside phosphonate with a mixed monothioacetal and thioaminal functionality is described. A 4-thiofuranosyl donor without 4’-hydroxymethyl group was used as scaffold for the introduction of a phosphonate moiety via Lewis-catalyzed glycosylation. After oxidation of the sulfur atom, the nucleobase uracil was introduced at the anomeric position using a Pummerer reaction, which led to the formation of a mixed monothioacetal and thioaminal functionality of l-4’-thiouridine phosphonate. This synthetic route provides valuable information to guide further synthetic efforts towards the synthesis of challenging nucleoside analogues such as l/d-4’-thionucleoside phosphonates.

Date:30 Nov 2017 →  7 Mar 2022
Keywords:Ribonucleotide Reductases Inhibitors
Disciplines:Microbiology, Systems biology, Laboratory medicine
Project type:PhD project