Elongation of N6-benzyladenosine scaffold via Pd-catalyzed C-C bond formation leads to derivatives with antiflaviviral activity.

Decoration of nucleoside analogues with lipophilic groups often leads to compounds with improved antiviral activity. For example, N6-benzyladenosine derivatives containing elongated lipophilic substituents in the benzyl core efficiently inhibit reproduction of tick-borne encephalitis virus (TBEV), while N6-benzyladenosine itself potently inhibits reproduction of human enterovirus A71 (EV-A71). We have extended a series of N6-benzyladenosine analogues using effective synthetic methods of CC bond formation based on Pd-catalyzed cross-coupling reactions (Sonogashira and Suzuki) in order to study the influence of bulky lipophilic substituents in the N6 position of adenosine on the antiviral activity against flaviviruses, such as TBEV, yellow fever virus (YFV) and West Nile virus (WNV), as well as a panel of enteroviruses including EV-A71, Echovirus 30 (E30), and poliovirus type 2 (PV2). Reproduction of tested flaviviruses appeared to be inhibited by the micromolar concentrations of the compounds, while cytotoxicity in most cases was beyond the detection limit. Time-of-addition studies demonstrated that the hit compounds inhibited the stage of viral RNA synthesis, but not the stages of the viral entry or protein translation. As a result, several new promising antiflaviviral leads have been identified. On the other hand, none of the synthesized compounds inhibited enterovirus reproduction, indicating a possibility of involvement of flavivirus-specific pathways in their mechanism of action.

Comparative Analysis of Enzymatic Transglycosylation Using E. coli Nucleoside Phosphorylases: A Synthetic Concept for the Preparation of Purine Modified 2'-Deoxyribonucleosides from Ribonucleosides

A comparative analysis of the transglycosylation conditions catalyzed by E. coli nucleoside phosphorylases, leading to the formation of 2'-deoxynucleosides, was performed. We demonstrated that maximal yields of 2'-deoxynucleosides, especially modified, can be achieved under small excess of glycosyl-donor (7-methyl-2'-deoxyguanosine, thymidine) and a 4-fold lack of phosphate. A phosphate concentration less than equimolar one allows using only a slight excess of the carbohydrate residue donor nucleoside to increase the reaction's output. A three-step methodology was elaborated for the preparative synthesis of purine-modified 2'-deoxyribonucleosides, starting from the corresponding ribonucleosides.

In Planta, In Vitro and In Silico Studies of Chiral N6-Benzyladenine Derivatives: Discovery of Receptor-Specific S-Enantiomers with Cytokinin or Anticytokinin Activities.

Cytokinins, classical phytohormones, affect all stages of plant ontogenesis, but their application in agriculture is limited because of the lack of appropriate ligands, including those specific for individual cytokinin receptors. In this work, a series of chiral N6-benzyladenine derivatives were studied as potential cytokinins or anticytokinins. All compounds contained a methyl group at the α-carbon atom of the benzyl moiety, making them R- or S-enantiomers. Four pairs of chiral nucleobases and corresponding ribonucleosides containing various substituents at the C2 position of adenine heterocycle were synthesized. A nucleophilic substitution reaction by secondary optically active amines was used. A strong influence of the chirality of studied compounds on their interaction with individual cytokinin receptors of Arabidopsis thaliana was uncovered in in vivo and in vitro assays. The AHK2 and CRE1/AHK4 receptors were shown to have low affinity for the studied S-nucleobases while the AHK3 receptor exhibited significant affinity for most of them. Thereby, three synthetic AHK3-specific cytokinins were discovered: N6-((S)-α-methylbenzyl)adenine (S-MBA), 2-fluoro,N6-((S)-α-methylbenzyl)adenine (S-FMBA) and 2-chloro,N6-((S)-α-methylbenzyl)adenine (S-CMBA). Interaction patterns between individual receptors and specific enantiomers were rationalized by structure analysis and molecular docking. Two other S-enantiomers (N6-((S)-α-methylbenzyl)adenosine, 2-amino,N6-((S)-α-methylbenzyl)adenosine) were found to exhibit receptor-specific and chirality-dependent anticytokinin properties.

In Vitro and In Silico Studies of Human Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) Inhibition by Stereoisomeric Forms of Lipophilic Nucleosides: The Role of Carbohydrate Stereochemistry in Ligand-Enzyme Interactions.

Inhibition of human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1) by different chiral lipophilic nucleoside derivatives was studied. New Tdp1 inhibitors were found in the series of the studied compounds with IC50 = 2.7-6.7 µM. It was shown that D-lipophilic nucleoside derivatives manifested higher inhibition activity than their L-analogs, and configuration of the carbohydrate moiety can influence the mechanism of Tdp1 inhibition.

Cytotoxicity reduction by O-nicotinoylation of antiviral 6-benzylaminopurine ribonucleosides.

One of the promising approaches in the development of nucleoside prodrugs is to use the nucleoside analogs containing lipophilic biodegradable residues, which are cleaved to biologically active forms after metabolic transformations in the cell. The introduction of such fragments makes it possible to reduce the general toxicity of the drug candidate and increase its stability in the cell. In order to study the influence of biodegradable lipophilic groups on antiviral activity and cytotoxicity, in this work we synthesized N6-benzyl-2',3',5'-tri-O-nicotinoyl adenosine and N6-(3-fluorobenzyl)-2',3',5'-tri-O-nicotinoyl adenosine, derivatives of N6-benzyladenosine (BAR) and N6-(3-fluorobenzyl)adenosine (FBAR), which had previously shown prominent antiviral activity against human enterovirus EV-A71 but appeared to be cytotoxic. The obtained fully-O-nicotinoylated BAR and FBAR inhibited reproduction of EV-A71 strains BrCr and 46973 and manifested significantly lower cytotoxicity compared to non-protected compounds. In addition, we performed enzymatic hydrolysis of the fully-O-nicotinoylated FBAR in the presence of esterases (CalB and PLE) to investigate metabolic degradation of O-nicotinoylated compounds in cells. Both enzymes hydrolyzed the tested substrate to form the corresponding O-deprotected nucleoside that may suggest the role of hydrolase-type enzymes as general participants of metabolic activation of O-nicotinoylated prodrugs in different cells.

Nucleoside Inhibitors of Coronaviruses.

Coronaviruses (CoVs) belong to a large family of zoonotic supercapsid viruses, including about 40 species of RNA-containing viruses with several strains capable of causing damage to the lungs and respiratory tract. The severe acute respiratory syndrome coronavirus (SARS-CoV) was responsible for the worldwide SARS outbreak in 2003. The rapid global spread of SARS-CoV-2 has been the cause of significant health concerns and thousands of deaths in 2019-2020 and outlined the need for novel antivirals. The present review is devoted to the development of effective and selective nucleoside drugs for the treatment of coronavirus infections. To date, about half of antivirals have been created based on nucleosides. The majority of drugs based on nucleosides have been approved by FDA. This indicates a fruitful area for the development of novel antivirals based on nucleosides. The review describes the main features of pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2 strains, presents their comparison, considers promising approaches to creating nucleoside drugs for the treatment of coronavirus infections and provides a systematic evaluation of all the known nucleoside derivatives, which inhibit the reproduction of coronaviruses in cells. To date, two known nucleoside drugs (Remdesivir, Favipiravir) have been recommended for the treatment of SARS-CoV-2 infection and nine hit compounds based on nucleosides and their analogues have been found, one of which efficiently suppressing SARS-CoV-2 replication and eight others inhibiting SARS-CoV replication.