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.

Inhibition of Tyrosyl-DNA Phosphodiesterase 1 by Lipophilic Pyrimidine Nucleosides.

Inhibition of DNA repair enzymes tyrosyl-DNA phosphodiesterase 1 and poly(ADP-ribose)polymerases 1 and 2 in the presence of pyrimidine nucleoside derivatives was studied here. New effective Tdp1 inhibitors were found in a series of nucleoside derivatives possessing 2',3',5'-tri-O-benzoyl-d-ribofuranose and 5-substituted uracil moieties and have half-maximal inhibitory concentrations (IC50) in the lower micromolar and submicromolar range. 2',3',5'-Tri-O-benzoyl-5-iodouridine manifested the strongest inhibitory effect on Tdp1 (IC50 = 0.6 µM). A decrease in the number of benzoic acid residues led to a marked decline in the inhibitory activity, and pyrimidine nucleosides lacking lipophilic groups (uridine, 5-fluorouridine, 5-chlorouridine, 5-bromouridine, 5-iodouridine, and ribothymidine) did not cause noticeable inhibition of Tdp1 (IC50 > 50 µM). No PARP1/2 inhibitors were found among the studied compounds (residual activity in the presence of 1 mM substances was 50-100%). Several O-benzoylated uridine and cytidine derivatives strengthened the action of topotecan on HeLa cervical cancer cells.

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases.

Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 5'-deoxy-5'-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the N↔S interconversion. In most of the complexes, the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn ↔ anti transitions. Such conformations of nucleosides in complexes are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme-substrate complex formation for this enzyme group.

Synthesis of Poly(ADP-ribose) Monomer Containing 2'-O-α-D-Ribofuranosyl Adenosine.

In this article, the earlier reported procedure for the synthesis of 2'-O-ß-D-ribofuranosyl nucleosides was extended to the synthesis of 2'-O-α-D-ribofuranosyl adenosine, a monomeric unit of poly(ADP-ribose). It consists in condensation of a small excess of 1-O-acetyl-2,3,5-tri-O-benzoyl-α,ß-D-arabinofuranose activated with tin tetrachloride with 3',5'-O-tetra-isopropyldisiloxane-1,3-diyl-ribonucleosides in 1,2-dichloroethane. The following debenzoylation and silylation of arabinofuranosyl residue and inversion of configuration at C-2'' atom of arabinofuranosyl residue and final removal of silyl protective groups gave 2'-O-α-D-ribofuranosyl adenosine in overall 13% to 21% yield. © 2019 by John Wiley & Sons, Inc.

A role for 3'-O-ß-D-ribofuranosyladenosine in altering plant immunity.

Our understanding of how, and the extent to which, phytopathogens reconfigure host metabolic pathways to enhance virulence is remarkably limited. Here we investigate the dynamics of the natural disaccharide nucleoside, 3'-O-ß-D-ribofuranosyladenosine, in leaves of Arabidopsis thaliana infected with virulent Pseudomonas syringae pv. tomato strain DC3000. 3'-O-ß-D-ribofuranosyladenosine is a plant derived molecule that rapidly accumulates following delivery of P. syringae type III effectors to represent a major component of the infected leaf metabolome. We report the first synthesis of 3'-O-ß-D-ribofuranosyladenosine using a method involving the condensation of a small excess of 1-O-acetyl-2,3,5-three-O-benzoyl-ß-ribofuranose activated with tin tetrachloride with 2',5'-di-O-tert-butyldimethylsilyladenosine in 1,2-dichloroethane with further removal of silyl and benzoyl protecting groups. Interestingly, application of synthetic 3'-O-ß-D-ribofuranosyladenosine did not affect either bacterial multiplication or infection dynamics suggesting a major reconfiguration of metabolism during pathogenesis and a heavy metabolic burden on the infected plant.

Synthesis of Cytokinins via Enzymatic Arsenolysis of Purine Nucleosides.

This unit describes an effective method for the preparation of natural cytokinins and their synthetic derivatives based on enzymatic cleavage of the N-glycosidic bond of N6 -substituted adenosine or O6 -substituted inosine derivatives in the presence of purine nucleoside phosphorylase (PNP) and Na2 HAsO4 . The arsenolysis reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, the desired products are formed in near-quantitative yields, as indicated by high-performance liquid chromatography (HPLC) analysis, and can easily be isolated. In the strategy used here, the ribose residue acts as a protective group. © 2018 by John Wiley & Sons, Inc.

Novel group of tyrosyl-DNA-phosphodiesterase 1 inhibitors based on disaccharide nucleosides as drug prototypes for anti-cancer therapy.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors based on disaccharide nucleosides was identified. TDP1 plays an essential role in the resistance of cancer cells to currently used antitumour drugs based on Top1 inhibitors such as topotecan and irinotecan. The most effective inhibitors investigated in this study have IC50 values (half-maximal inhibitory concentration) in 0.4-18.5 µM range and demonstrate relatively low own cytotoxicity along with significant synergistic effect in combination with anti-cancer drug topotecan. Moreover, kinetic parameters of the enzymatic reaction and fluorescence anisotropy were measured using different types of DNA-biosensors to give a sufficient insight into the mechanism of inhibitor's action.

Synthesis of N6 -Substituted Adenosines as Cytokinin Nucleosides.

This unit describes preparation of N6 -substituted adenosines (cytokinin nucleosides), a unique class of compounds with a wide spectrum of biological activities. Regioselective alkylation of N6 -acetyl-2',3',5'-tri-O-acetyladenosine with alkyl halides under basic conditions or alcohols under Mitsunobu conditions followed by deprotection are the methods of choice for the preparation of the cytokinin nucleosides. The attractive feature of this strategy is the possibility of using a broad library of commercially available alkyl halides and alcohols under mild reaction conditions. © 2018 by John Wiley & Sons, Inc.

Chemoenzymatic synthesis of cytokinins from nucleosides: ribose as a blocking group.

Nucleoside phosphorylases are involved in the salvage pathways of nucleoside biosynthesis and catalyze the reversible reaction of a nucleobase with α-d-ribose-1-phosphate to yield a corresponding nucleoside and an inorganic phosphate. The equilibrium of these reactions is shifted towards nucleosides, especially in the case of purines. Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is widely used in labs and industry for the synthesis of nucleosides of practical importance. Bacterial PNPs have relatively broad substrate specificity utilizing a wide range of purines with different substituents to form the corresponding nucleosides. To shift the reaction in the opposite direction we have used arsenolysis instead of phosphorolysis. This reaction is irreversible due to the hydrolysis of the resulting α-d-ribose-1-arsenate. As a result, heterocyclic bases are formed in quantitative yields and can be easily isolated. We have developed a novel method for the preparation of cytokinins based on the enzymatic cleavage of the N-glycosidic bond of N6-substituted adenosines in the presence of PNP and Na2HAsO4. According to the HPLC analysis the conversion proceeds in quantitative yields. In the proposed strategy the ribose residue acts as a protective group. No contamination of the final products with AsO43- has been detected via HPLC-HRMS; simple analytical arsenate detection via ESI-MS has been proposed.

Fluorination of Naturally Occurring N6-Benzyladenosine Remarkably Increased Its Antiviral Activity and Selectivity.

Recently, we demonstrated that the natural cytokinin nucleosides N6-isopentenyladenosine (iPR) and N6-benzyladenosine (BAPR) exert a potent and selective antiviral effect on the replication of human enterovirus 71. In order to further characterize the antiviral profile of this class of compounds, we generated a series of fluorinated derivatives of BAPR and evaluated their activity on the replication of human enterovirus 71 in a cytopathic effect (CPE) reduction assay. The monofluorination of the BAPR-phenyl group changed the selectivity index (SI) slightly because of the concomitant high cell toxicity. Interestingly, the incorporation of a second fluorine atom resulted in a dramatic improvement of selectivity. Moreover, N6-trifluoromethylbenzyladenosines derivatives (9-11) exhibited also a very interesting profile, with low cytotoxicity observed. In particular, the analogue N6-(3-trifluoromethylbenzyl)-adenosine (10) with a four-fold gain in potency as compared to BAPR and the best SI in the class represents a promising candidate for further development.

New tools in nucleoside toolbox of tick-borne encephalitis virus reproduction inhibitors.

Design and development of nucleoside analogs is an established strategy in the antiviral drug discovery field. Nevertheless, for many viruses the coverage of structure-activity relationships (SAR) in the nucleoside chemical space is not sufficient. Here we present the nucleoside SAR exploration for tick-borne encephalitis virus (TBEV), a member of Flavivirus genus. Promising antiviral activity may be achieved by introduction of large hydrophobic substituents in the position 6 of adenosine or bulky silyl groups to the position 5'. Introduction of methyls to the ribose moiety does not lead to inhibition of TBEV reproduction. Possible mechanisms of action of these nucleosides include the inhibition of viral entry or interaction with TBEV non-structural protein 5 methyltransferase or RNA-dependent RNA polymerase domains.

Anti-HIV Activities of Intramolecular G4 and Non-G4 Oligonucleotides.

New natural and chemically modified DNA aptamers that inhibit HIV-1 activity at submicromolar concentrations (presumably via preventing viral entry into target cells) are reported. The new DNA aptamers were developed based on known intramolecular G-quadruplexes (G4s) that were functionally unrelated to HIV inhibition [the thrombin-binding aptamer and the fragment of the human oncogene promoter (Bcl2)]. The majority of previously described DNA inhibitors of HIV infection adopt intermolecular structures, and thus their folding variability represents an obvious disadvantage. Intramolecular architectures refold correctly after denaturation and are generally easier to handle. However, whether the G4 topology or other factors account for the anti-HIV activity of our aptamers is unknown. The impact of chemical modification (thiophosphoryl internucleotide linkages) on aptamer activity is discussed. The exact secondary structures of the active compounds and further elucidation of their mechanisms of action hopefully will be the subjects of future studies.

Poly(ADP-ribose): From chemical synthesis to drug design.

Poly(ADP-ribose) (PAR) is an important biopolymer, which is involved in various life processes such as DNA repair and replication, modulation of chromatin structure, transcription, cell differentiation, and in pathogenesis of various diseases such as cancer, diabetes, ischemia and inflammations. PAR is the most electronegative biopolymer and this property is essential for its binding with a wide range of proteins. Understanding of PAR functions in cell on molecular level requires chemical synthesis of regular PAR oligomers. Recently developed methodologies for chemical synthesis of PAR oligomers, will facilitate the study of various cellular processes, involving PAR.

Cytokinin Nucleosides - Natural Compounds with a Unique Spectrum of Biological Activities.

Cytokinin nucleosides exhibit antitumor, antiviral, antiprotozoal, blood pressure reducing, anti-inflammatory, and antipsychotic activity. These compounds also influence platelet aggregation and exhibit some other biological activities. Cytokinins are N6-substituted adenines and represent an important group of phytohormones with diverse biochemical functions in plants, stimulating cell division and plant growth. The main structural feature of cytokinin nucleosides is the presence of a hydrophobic hydrocarbon moiety at the N6-position of adenosine. This moiety is responsible for a difference in physicochemical and biological properties as compared to adenosine. 1-N-Tuberculosinyladenosine and N6-tuberculosinyladenosine are specifically produced by Mycobacterium tuberculosis as components of the plasmatic membrane, thus making them attractive targets for clinical test development. Structurally related compounds were found in marine organisms. It has been shown also that tRNA contains N6-isoprenyladenosine and some other related compounds. This review summarizes the structural features, biological activity, and the synthesis of cytokinin nucleosides and some of their closely related derivatives such as cytokinins and terpene derivatives of adenine.

Modification of the length and structure of the linker of N(6)-benzyladenosine modulates its selective antiviral activity against enterovirus 71.

Very recently, we demonstrated that N(6)-isopentenyladenosine, a cytokinin nucleoside, exerts a potent and selective antiviral effect on the replication of human enterovirus 71. The present study is devoted to the structure optimization of another natural compound: N(6)-benzyladenosine. We mainly focused on the exploration of the size and nature of the linker between the adenine and the phenyl ring, as well as on the necessity of the D-ribose residue. More than 30 analogues of N(6)-benzyladenosine were prepared and their antiviral properties were evaluated. Two main methodologies were used for preparation: N(6)-acetyl-2',3',5'-tri-O-acetyladenosine can be regioselectively alkylated either by alkyl halides under base promoted conditions or by alcohols in Mitsunobu reactions. After deacylation with 4 M PrNH2 in MeOH at room temperature for one day, the desired products were obtained in overall high yields. Analysis of the structure-activity relationship clearly shows that the optimal size of the linker is limited to 2 or 3 atoms (compounds 4-7). 2'-Deoxyadenosine derivatives did not elicit any inhibitory or cytotoxic effect, while 5'-deoxynucleosides still induced some cell protective antiviral activity. Based on these observations, it can be hypothesized that there may be another mechanism that is at the base of the antiviral activity of these compounds against enterovirus 71 besides a possible 5'-triphosphorylation followed by a putative inhibitory effect on RNA synthesis.

Crosslinking of Chitosan with Dialdehyde Derivatives of Nucleosides and Nucleotides. Mechanism and Comparison with Glutaraldehyde.

In medical and pharmaceutical applications, chitosan is used as a component of hydrogels-macromolecular networks swollen in water. Chemical hydrogels are formed by covalent links between the crosslinking reagents and amino functionalities of chitosan. To date, the most commonly used chitosan crosslinkers are dialdehydes, such as glutaraldehyde (GA). We have developed novel GA like crosslinkers with additional functional groups-dialdehyde derivatives of uridine (oUrd) and nucleotides (oUMP and oAMP)-leading to chitosan-based biomaterials with new properties. The process of chitosan crosslinking was investigated in details and compared to crosslinking with GA. The rates of crosslinking with oUMP, oAMP, and GA were essentially the same, though much higher than in the case of oUrd. The remarkable difference in the crosslinking properties of nucleoside and nucleotide dialdehydes can be clearly attributed to the presence of the phosphate group in nucleotides that participates in the gelation process through ionic interactions with the amino groups of chitosan. Using NMR spectroscopy, we have not observed the formation of aldimine bonds. It can be concluded that the real number of crosslinks needed to cause gelation of chitosan chains may be less than 1%.

Regioselective 1-N-Alkylation and Rearrangement of Adenosine Derivatives.

Several methods for the preparation of some N(6)-substituted adenosines based on selective 1-N-alkylation with subsequent Dimroth rearrangement were developed. The proposed methods seem to be effective for the preparation of natural N(6)-isopentenyl- and N(6)-benzyladenosines, which are known to possess pronounced biological activities. Direct 1-N-alkylation of 2',3',5'-tri-O-acetyladenosine and 3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides in N,N-dimethylformamide (DMF) in the presence of BaCO3 and KI gave 1-N-substituted derivatives with quantitative yields, whereas 1-N-alkylation of adenosine was accompanied by significant O-alkylation. Moreover, the reaction of trimethylsilyl derivatives of N(6)-acetyl-2',3',5'-tri-O-acetyladenosine and N(6)-acetyl-3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides leads to the formation of the stable 1-N-substituted adenosines. Dimroth rearrangement of 1-N-substituted adenosines in aqueous ammonia yields pure N(6)-substituted adenosines.

Poly(ADP-ribose)--a unique natural polymer structural features, biological role and approaches to the chemical synthesis.

Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them, poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective target for the design of new drugs, affecting PAR metabolism. The structure of PAR was established by enzymatic hydrolysis and further analysis of the products, but total chemical synthesis of PAR hasn't been described yet. Several approaches have been developed on the way to chemical synthesis of this unique biopolymer.

Chemical modification of the plant isoprenoid cytokinin N(6)-isopentenyladenosine yields a selective inhibitor of human enterovirus 71 replication.

In this study, we demonstrate that N(6)-isopentenyladenosine, which essentially is a plant cytokinin-like compound, exerts a potent and selective antiviral effect on the replication of human enterovirus 71 with an EC50 of 1.0 ± 0.2 µM and a selectivity index (SI) of 5.7. The synthesis of analogs with modification of the N(6)-position did not result in a lower EC50 value. However, in particular with the synthesis of N(6)-(5-hexene-2-yne-1-yl)adenosine (EC50 = 4.3 ± 1.5 µM), the selectivity index was significantly increased: because of a reduction in the adverse effect of this compound on the host cells, an SI > 101 could be calculated. With this study, we for the first time provide proof that a compound class that is based on the plant cytokinin skeleton offers an interesting starting point for the development of novel antivirals against mammalian viruses, in the present context in particular against enterovirus 71.

Disaccharide pyrimidine nucleosides and their derivatives: a novel group of cell-penetrating inhibitors of poly(ADP-ribose) polymerase 1.

Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3'-O-ß-D-ribofuranosyl-5-iodo-dUrd (2d; IC50 = 45 µM), 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine (2e; IC50 = 38 µM), and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine oxidized (4; IC50 = 25 µM). These compounds also reduced H2O2-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.