Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA.

The SARS-CoV-2 betacoronavirus pandemic has claimed more than 6.5 million lives and, despite the development and use of COVID-19 vaccines, remains a major global public health problem. The development of specific drugs for the treatment of this disease remains a very urgent task. In the context of a repurposing strategy, we previously screened a library of nucleoside analogs showing different types of biological activity against the SARS-CoV-2 virus. The screening revealed compounds capable of inhibiting the reproduction of SARS-CoV-2 with EC50 values in the range of 20-50 µM. Here we present the design and synthesis of various analogs of the leader compounds, the evaluation of their cytotoxicity and antiviral activity against SARS-CoV-2 in cell cultures, as well as experimental data on RNA-dependent RNA polymerase inhibition. Several compounds have been shown to prevent the interaction between the SARS-CoV-2 RNA-dependent RNA polymerase and the RNA substrate, likely inhibiting virus replication. Three of the synthesized compounds have also been shown to inhibit influenza virus. The structures of these compounds can be used for further optimization in order to develop an antiviral drug.

New Flexible Analogues of 8-Aza-7-deazapurine Nucleosides as Potential Antibacterial Agents.

A variety of ribo-, 2'-deoxyribo-, and 5'-norcarbocyclic derivatives of the 8-aza-7-deazahypoxanthine fleximer scaffolds were designed, synthesized, and screened for antibacterial activity. Both chemical and chemoenzymatic methods of synthesis for the 8-aza-7-deazainosine fleximers were compared. In the case of the 8-aza-7-deazahypoxanthine fleximer, the transglycosylation reaction proceeded with the formation of side products. In the case of the protected fleximer base, 1-(4-benzyloxypyrimidin-5-yl)pyrazole, the reaction proceeded selectively with formation of only one product. However, both synthetic routes to realize the fleximer ribonucleoside (3) worked with equal efficiency. The new compounds, as well as some 8-aza-7-deazapurine nucleosides synthesized previously, were studied against Gram-positive and Gram-negative bacteria and M. tuberculosis. It was shown that 1-(ß-D-ribofuranosyl)-4-(2-aminopyridin-3-yl)pyrazole (19) and 1-(2',3',4'-trihydroxycyclopent-1'-yl)-4-(pyrimidin-4(3H)-on-5-yl)pyrazole (9) were able to inhibit the growth of M. smegmatis mc2 155 by 99% at concentrations (MIC99) of 50 and 13 µg/mL, respectively. Antimycobacterial activities were revealed for 4-(4-aminopyridin-3-yl)-1H-pyrazol (10) and 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-4-(4-benzyloxypyrimidin-5-yl)pyrazole (6). At concentrations (MIC99) of 40 and 20 µg/mL, respectively, the compounds resulted in 99% inhibition of M. tuberculosis growth.

Design and Synthesis of New Modified Flexible Purine Bases as Potential Inhibitors of Human PNP.

The great interest in studying the structure of human purine nucleoside phosphorylase (hPNP) and the continued search for effective inhibitors is due to the importance of the enzyme as a target in the therapy of T-cell proliferative diseases. In addition, hPNP inhibitors are used in organ transplant surgeries to provide immunodeficiency during and after the procedure. Previously, we showed that members of the well-known fleximer class of nucleosides are substrates of E. coli PNP. Fleximers have great promise as they have exhibited significant biological activity against a number of viruses of pandemic concern. Herein, we describe the synthesis and inhibition studies of a series of new fleximer compounds against hPNP and discuss their possible binding mode with the enzyme. At a concentration of 2 mM for the flex-7-deazapurines 1-4, a decrease in enzymatic activity by more than 50% was observed. 4-Amino-5-(1H-pyrrol-3-yl)pyridine 2 was the best inhibitor, with a Ki = 0.70 mM. Docking experiments have shown that ligand 2 is localized in the selected binding pocket Glu201, Asn243 and Phe200. The ability of the pyridine and pyrrole fragments to undergo rotation around the C-C bond allows for multiple binding modes in the active site of hPNP, which could provide several plausible bioactive conformations.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity.

Pyrimidine nucleoside analogues are widely used to treat infections caused by the human immunodeficiency virus (HIV) and DNA viruses from the herpes family. It has been shown that 5-substituted uracil derivatives can inhibit HIV-1, herpes family viruses, mycobacteria and other pathogens through various mechanisms. Among the 5-substituted pyrimidine nucleosides, there are not only the classical nucleoside inhibitors of the herpes family viruses, 2'-deoxy-5-iodocytidine and 5-bromovinyl-2'-deoxyuridine, but also derivatives of 1-(benzyl)-5-(phenylamino)uracil, which proved to be non-nucleoside inhibitors of HIV-1 and EBV. It made this modification of nucleoside analogues very promising in connection with the emergence of new viruses and the crisis of drug resistance when the task of creating effective antiviral agents of new types that act on other targets or exhibit activity by other mechanisms is very urgent. In this paper, we present the design, synthesis and primary screening of the biological activity of new nucleoside analogues, namely, 5'-norcarbocyclic derivatives of substituted 5-arylamino- and 5-aryloxyuracils, against RNA viruses.

Novel fleximer pyrazole-containing adenosine analogues: chemical, enzymatic and highly efficient biotechnological synthesis.

Nucleoside analogues have long served as key chemotherapeutic drugs for the treatment of viral infections and cancers. Problems associated with the development of drug resistance have led to a search for the design of nucleosides capable of bypassing point mutations in the target enzyme's binding site. As a possible answer to this, the Seley-Radtke group developed a flexible nucleoside scaffold (fleximers), where the heterocyclic purine base is split into its two components, i.e. pyrimidine and imidazole. Herein, we present a series of new pyrazole-containing flex-bases and the corresponding fleximer analogues of 8-aza-7-deaza nucleosides. Subsequent studies found that pyrazole-containing flex-bases are substrates of purine nucleoside phosphorylase (PNP). We have compared the chemical synthesis of fleximers and enzymatic approaches with both isolated enzymes and the use of E. coli cells overproducing PNP. The latter provided stereochemically pure pyrazole-containing ß-d-ribo- and ß-d-2'-deoxyribo-fleximers and are beneficial in terms of environmental issues, are more economical, and streamline the steps required from a chemical approach. The reaction is carried out in water, avoiding hazardous chemicals, and the products are isolated by ion-exchange chromatography using water/ethanol mixtures for elution. Moreover, the target nucleosides were obtained on a multi-milligram scale with >97-99% purity, and the reactions can be easily scaled up.

Uracil-Containing Heterodimers of a New Type: Synthesis and Study of Their Anti-Viral Properties.

Widespread latent herpes viral infections within a population can lead to the development of co-infections in HIV-infected patients. These infections are not particularly dangerous for healthy individuals and often occur with minimal symptoms, but for those who are immunocompromised, these infections can accelerate the acute phase of HIV infection and AIDS. Thus, the idea of designing compounds that could combine activity against HIV and co-infections would seem promising. In that regard, eleven compounds were synthesized that represent conjugates of non-nucleoside HIV reverse transcriptase inhibitors and nucleoside inhibitors of the herpes family viruses with the hope that these novel heterodimers will result in dual activity against HIV and concomitant herpes virus infections.

Investigation of 5'-Norcarbocyclic Nucleoside Analogues as Antiprotozoal and Antibacterial Agents.

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.

Novel 5'-Norcarbocyclic Derivatives of Bicyclic Pyrrolo- and Furano[2,3-d]Pyrimidine Nucleosides.

Here we report the synthesis and biological activity of new 5'-norcarbocyclic derivatives of bicyclic pyrrolo- and furano[2,3-d]pyrimidines with different substituents in the heterocyclic ring. Lead compound 3i, containing 6-pentylphenyl substituent, displays inhibitory activity with respect to a number of tumor cells with a moderate selectivity index value. Compound 3i induces cell death by the apoptosis pathway with the dissipation of mitochondrial potential.

Novel 5'-Norcarbocyclic Pyrimidine Derivatives as Antibacterial Agents.

A series of novel 5'-norcarbocyclic derivatives of 5-alkoxymethyl or 5-alkyltriazolyl-methyl uracil were synthesized and the activity of the compounds evaluated against both Gram-positive and Gram-negative bacteria. The growth of Mycobacterium smegmatis was completely inhibited by the most active compounds at a MIC99 of 67 µg/mL (mc²155) and a MIC99 of 6.7⁻67 µg/mL (VKPM Ac 1339). Several compounds also showed the ability to inhibit the growth of attenuated strains of Mycobacterium tuberculosis ATCC 25177 (MIC99 28⁻61 µg/mL) and Mycobacterium bovis ATCC 35737 (MIC99 50⁻60 µg/mL), as well as two virulent strains of M. tuberculosis; a laboratory strain H37Rv (MIC99 20⁻50 µg/mL) and a clinical strain with multiple drug resistance MS-115 (MIC99 20⁻50 µg/mL). Transmission electron microscopy (TEM) evaluation of M. tuberculosis H37Rv bacterial cells treated with one of the compounds demonstrated destruction of the bacterial cell wall, suggesting that the mechanism of action for these compounds may be related to their interactions with bacteria cell walls.