Requirements for novel effective antiviral agents against SARS-CoV-2 emphasizes the importance of robust in vitro screening platforms. We developed a test system based on spike-pseudotyped lentiviruses, carrying either luc+ or EGFP reporter genes as a payload, and a human non-small cell lung carcinoma (NSCLC) cell line, overexpressing ACE2 (H1299-hACE2). The cell origin makes our system resemble lung epithelium infection. Transmission electron microscopy confirmed that the spike glycoproteins on the pseudotyped lentiviral particles resemble native SARS-CoV-2 spike glycoproteins, thus validating their use in inhibitor screening. H1299-hACE2 cells showed significantly higher infection rate (p < 0.005) with spike-pseudotyped lentiviruses compared to parental H1299 cells, as determined by luciferase and fluorescence assays. The susceptibility of the stable H1299-hACE2 cell line to a broad panel of SARS-CoV-2 variants (Wuhan, Beta, Delta, Omicron) was assessed here for the first time in a unified experimental setting. Infection of H1299-hACE2 cells with SARS-CoV-2 induced cell fusion and syncytium formation with subsequent cell death. The developed pseudovirus-based assay was further used for assessment of the antiviral properties of derivatives of 1,7,7-trimethyl-[2.2.1]-bicycloheptane-potential spike protein inhibitors, which possess moderate activity against lentiviral particles. The H1299-hACE2/spike-pseudotyped lentivirus assay is, therefore, a reliable, high-efficiency platform for screening spike-mediated entry inhibitors. The cell line obtained during the development of the platform can be used to isolate and study new variants of SARS-CoV-2.

Allobetulin derivatives with rearranged E-ring exhibit important pharmacological properties against various viral pathogens. Basing on 3β-acetoxy-21β-acetyl-20β,28-epoxy-18α,19βH-ursane triterpenoid, obtained from allobetulin in one stage, a series of new derivatives have been synthesized and their antiviral activity against both influenza virus A (H1N1) and SARS-CoV-2 pseudovirus was evaluated. Among them, 2,3-indolo-allobetulone N-propargyl derivatives were the most efficacious against influenza virus with low toxicity (CC50 × 300 μM) and IC50 values of 7.04 to 3.5 μM and high selectivity indexes (SI 43 and 86). Compared with amodiaquine, a derivative with 3-pyridinylidene fragment 12 showed a weak antiviral activity against SARS-CoV-2 pseudovirus with an inhibition rate of 57.3

A series of new dimers of pyrimidine nucleoside analogues have been synthesized. The dimers are composed of two uracil or thymine fragments linked at N3 through a polymethylene bridge and bearing a β-D-ribofuranose residue linked to N1 of the nucleobase through a 1,2,3-triazolylalkyl spacer. Biological screening revealed that some of the synthesized dimers are active against influenza A (H1N1) virus and coxsackievirus B3 with IC50 values of 13 and 4.5 μM, respectively.

Based on the fact that a search for influenza antivirals among nucleoside analogues has drawn very little attention of chemists, the present study reports the synthesis of a series of 1,2,3-triazolyl nucleoside analogues in which a pyrimidine fragment is attached to the ribofuranosyl-1,2,3-triazol-4-yl moiety by a polymethylene linker of variable length. Target compounds were prepared by the Cu alkyne-azide cycloaddition (CuAAC) reaction. Derivatives of uracil, 6-methyluracil, 3,6-dimethyluracil, thymine and quinazolin-2,4-dione with ω-alkyne substituent at the N1 (or N5) atom and azido 2,3,5-tri-O-acetyl-D-β-ribofuranoside were used as components of the CuAAC reaction. All compounds synthesized were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1) and coxsackievirus B3. The best values of IC50 (inhibiting concentration) and SI (selectivity index) were demonstrated by the lead compound 4i in which the 1,2,3-triazolylribofuranosyl fragment is attached to the N1 atom of the quinazoline-2,4-dione moiety via a butylene linker (IC50 = 30 μM, SI = 24) and compound 8n in which the 1,2,3-triazolylribofuranosyl fragment is attached directly to the N5 atom of the 6-methyluracil moiety (IC50 = 15 μM, SI = 5). According to theoretical calculations, the antiviral activity of the 1,2,3-triazolyl nucleoside analogues 4i and 8n against H1N1 (A/PR/8/34) influenza virus can be explained by their influence on the functioning of the polymerase acidic protein (PA) of RNA-dependent RNA polymerase (RdRP).

llobetulin derivatives with rearranged E -ring exhibit important pharmacological properties against various viral pathogens. Basing on 3 β -acetoxy-21 β -acetyl-20 β ,28-epoxy-18 α ,19 βH -ursane triterpenoid, obtained from allobetulin in one stage, a series of new derivatives have been synthesized and their antiviral activity against both influenza virus A (H1N1) and SARS-CoV-2 pseudovirus was evaluated. Among them, 2,3-indolo-allobetulone N -propargyl derivatives were the most efficacious against influenza virus with low toxicity (CC 50 × 300 μM) and IC 50 values of 7.04 to 3.5 μM and high selectivity indexes (SI 43 and 86). Compared with amodiaquine, a derivative with 3-pyridinylidene fragment 12 showed a weak antiviral activity against SARS-CoV-2 pseudovirus with an inhibition rate of 57.3% at a concentration of 20 μM. Further optimization to increase the cellular antiviral activity seems to be necessary to develop this series of triterpenoids as antiviral agents.

Herein, we present the first experimental study of individual water-soluble fullerene derivatives proving their ability to inhibit SARS-CoV-2 in vitro. The initial screening allowed us to identify a few new compounds that have demonstrated pronounced antiviral activity with IC50 values as low as 390 nM and selectivity indexes reaching 214. Time-of-addition analysis and molecular docking results suggested that the viral protease and/or the spike protein are the most probable targets inhibited by the fullerene derivatives. Further rational design of fullerene derivatives might lead to the development of compounds with further enhanced antiviral activity and decreased toxicity.

A set of triterpene A-ring hydroxymethylene-amino-derivatives was synthesized and their antiviral activity was studied. The synthesized compounds were tested for their potential inhibition of SARS-CoV-2 pseudovirus in BHK-21-hACE2 cells and influenza A/PuertoRico/8/34 (H1N1) virus in MDCK cell culture. Compounds 6 , 8 and 19 showed significant anti-SARS-CoV-2 pseudovirus activity with EC 50 value of 3.20-11.13 µM, which is comparable to the positive control amodiaquine (EC 50 3.17 µM). Among them, 28- O -imidazolyl-azepano-betulin 6 and C3-hydroxymethylene-amino-glycyrrhetol-11,13 - diene 19 were identified as the lead compounds with SI values of 7 and 10. The binding mode of compound 6 into the RBD domain of SARS-CoV-2 spike glycoprotein (PDB code: 7DK3) by docking and molecular dynamics simulation was investigated.

By covalent binding of 1,2,3-triazolyl nucleoside analogues by a polymethylene chain at the N3 atoms, a series of homodimers was synthesized, in which nucleic bases are replaced by quinazoline-2,4-dione moieties connected to the β-D-ribofuranose residues by 1,2,3-triazolylbutyl linkers. Screening of in vitro antiviral activity against influenza A (H1N1) and Coxsackie B3 viruses revealed a lead compound that showed high antiviral activity (IC50 = 11.6 μM) against influenza A/Puerto Rico/8/34 (H1N1) virus and a lead compound that exhibited high antiviral activity (IC50 = 12.2 μM) against enterovirus Coxsackie B3.

Introduction . The requirements of modern clinical guidelines for the treatment of respiratory viral infections suggest the possibility of identifying a specific viral pathogen. In this regard, the search for drugs with selective activity against respiratory syncytial virus and parainfluenza virus is relevant. The purpose of the work is to study the antiviral activity of the drug Cytovir®-3 in vitro in relation to the cytopathogenic effect of respiratory viruses (parainfluenza virus and respiratory syncytial virus). Material and methods . The antiviral effect of Cytovir®-3 in comparison with Umifenovir against parainfluenza virus and respiratory syncytial virus was studied on Vero cell culture. Drugs were administered 1 hour before (prophylactic regimen) and 1 hour after (treatment regimen) infection of the cell culture with respiratory viruses. The working range of concentrations of the studied drugs was calculated based on the values of 50% cytotoxic concentration calculated based on the results of a quantitative microtetrazole test. Results and discussion. The drug Cytovir®-3 in two schemes of application (therapeutic or prophylactic) showed its antiviral efficacy in vitro against respiratory syncytial virus and parainfluenza virus due in the non-toxic range (0–794 µg/ml). At the same time, the suppressive effect of the drug Cytovir®-3 against the parainfluenza virus begins at lower concentrations of the drug with its early (preventive) introduction into cell culture (250 mcg/ml). Conclusion . Antiviral activity of Cytovir®-3 has been proven in vitro against parainfluenza virus and respiratory syncytial virus. At the same time, in all series of experiments, Cytovir®-3 had a higher index of selectivity of antiviral action than that of the comparison drug Umifenovir.

A comparative analysis of in vitro antiviral activity (in terms of the concentration of semi-maximal inhibition, IC50) against influenza virus A/PR/8/34 (H1N1) of a large series of parent 1,2,3-triazolyl nucleoside analogues (with uracil, thymine, 6-methyluracil, quinazoline-2,4-dione moieties as nucleic bases) and their prodrug forms with masked 5ʹ-phosphate groups (diethyl phosphate, diphenyl phosphate, phosphoramidate) and negatively charged H-phosphonate and monophosphate groups was carried out. Obtained structure-activity relationships were interpreted based on the assumption that the synthesized parent 1,2,3-triazolyl nucleoside analogues and their prodrug forms, by analogy with the literature data, are metabolized by cellular kinases to their active 5ʹ-triphosphate forms that inhibit the activity of viral RNA-dependent RNA polymerase (RdRp). A correlation was found between the experimental values of IC50 and the theoretical values of the binding energies of 5ʹ-triphosphate derivatives of the parent 1,2,3-triazolyl nucleoside analogues in the active site of RdRp.

Oleanolic and glycyrrhetic acids alkyne derivatives were synthesized as a result of propargylation of the indole NH-group condensed with the triterpene A-ring, the following aminomethylation led to a series of Mannich bases. The synthesized compounds were tested for their potential inhibition of influenza A/PuertoRico/8/34 (H1N1) virus in Madin-Darby canine kidney (MDCK) cell culture and SARS-CoV-2 pseudovirus in baby hamster kidney-21-human angiotensin-converting enzyme 2 (BHK-21-hACE2) cells. Mannich bases of oleanolic and glycyrrhetic acids N-propargylated indoles 7, 8, and 12 were the most efficacious against influenza virus A with IC50 7-10 μM together with a low toxicity (CC50  > 145 μM) and high selectivity index SI value 20. Indolo-oleanolic acid morpholine amide Mannich base holding N-methylpiperazine moiety 9 showed anti-SARS-CoV-2 pseudovirus activity with EC50 value of 14.8 μM. Molecular docking and dynamics modeling investigated the binding mode of the compounds 7 and 12 into the binding pocket of influenza A virus M2 protein and compound 9 into the RBD domain of SARS-CoV-2 spike glycoprotein.

A series of 2,4-disubstituted pyrrolo[2,1-f][1,2,4]triazines containing both aryl and thienyl substituents were synthesized by exploiting the 1,3-cycloaddition reaction of N(1)-ethyl-1,2,4-triazinium tetrafluoroborates with dimethyl acetylenedicarboxylate. The antiviral activity of the synthesized compounds against influenza virus strain A/Puerto Rico/8/34 (H1N1) was studied in experiments on Madin-Darby canine kidney (MDCK) cell culture. Among the pyrrolo[2,1-f][1,2,4]triazine derivatives, compounds with low toxicity and high antiviral activity were identified. Dimethyl 4-(4-methoxyphenyl)-7-methyl-2-p-tolylpyrrolo[2,1-f][1,2,4]triazine-5,6-dicarboxylate was found to demonstrate the best antiviral activity (IC50 4 µg/mL and selectivity index 188). Based on the results of in vitro tests and molecular docking studies performed, a plausible mechanism of action for these compounds was suggested to involve inhibition of neuraminidase.

Interaction of 2-furylvinyl-and 2-thienylvinyl-2,3-dihydroquinazolin-4(1H)-ones with maleic anhydride gives isoindolo[2,1-a]quinazoline-11(13)-carboxylic acids via the tandem acylation/intramolecular Diels-Alder reaction of vinylarenes (IMDAV). The reaction sequence involves three successive steps: the initial acylation of a quinazolinone nitrogen atom, the IMDAV reaction, and the final aromatization of the resulting five-member ring. The proposed exo-cycloaddition reaction allows to construct from four to five stereogenic centers and two new rings in one synthetic step. The target annelated isoindoloquinazolines were evaluated in vitro against common bacterial pathogens strains (Escherichia coli, Staphylococcus aureus, etc.). It was demonstrated that some of thienoisoindolequinazoline derivatives exibit a potent antibacterial activity at the level of MIC values of 16-32 mg mL 1. Selected 2,3-dihydroquinazolin-4(1H)-ones and isoindolo[2,1-a]quinazolinecarboxylic acids displayed antiviral activity (against the influenza virus A/Puerto Rico/8/34 -H1N1).(c) 2022 Elsevier Ltd. All rights reserved.

A series of 1,2,3-triazolyl nucleoside analogues bearing N-acetyl-D-glucosamine residue was synthesized by the copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction of N1-omega-alkynyl derivatives of uracil, 6-methyluracil, thymine and 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-beta-D-glucopyranosyl azide. Antiviral assays revealed the lead compound 3f which showed both the same activity against the influenza virus A H1N1 (IC50=70.7 mu M) as the antiviral drug Rimantadine in control (IC50=77 mu M) and good activity against Coxsackievirus B3 (IC50=13.9 mu M) which was one and a half times higher than the activity of the antiviral drug Pleconaril in control (IC50=21.6 mu M). According to molecular docking simulations, the antiviral activity of the lead compound 3f against Coxsackie B3 virus can be explained by its binding to a key fragment of the capsid surface of this virus.

With the resurgence of the coronavirus pandemic, the repositioning of FDA-approved drugs against coronovirus and finding alternative strategies for antiviral therapy are both important. We previously identified the viral lipid envelope as a potential target for the prevention and treatment of SARS-CoV-2 infection with plant alkaloids (Shekunov et al., 2021). Here, we investigated the effects of eleven cyclic lipopeptides (CLPs), including well-known antifungal and antibacterial compounds, on the liposome fusion triggered by calcium, polyethylene glycol 8000, and a fragment of SARS-CoV-2 fusion peptide (816-827) by calcein release assays. Differential scanning microcalorimetry of the gel-to-liquid-crystalline and lamellar-to-inverted hexagonal phase transitions and confocal fluorescence microscopy demonstrated the relation of the fusion inhibitory effects of CLPs to al-terations in lipid packing, membrane curvature stress and domain organization. The antiviral effects of CLPs were evaluated in an in vitro Vero-based cell model, and aculeacin A, anidulafugin, iturin A, and mycosubtilin attenuated the cytopathogenicity of SARS-CoV-2 without specific toxicity.

A series of 5′-phosphorylated (dialkyl phosphates, diaryl phosphates, phosphoramidates, H-phosphonates, phosphates) 1,2,3-triazolyl nucleoside analogues in which the 1,2,3-triazole-4-yl-β-D-ribofuranose fragment is attached via a methylene group or a butylene chain to the N-1 atom of the heterocycle moiety (uracil or quinazoline-2,4-dione) was synthesized. All compounds were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1). Antiviral assays revealed three compounds, 13b, 14b, and 17a, which showed moderate activity against influenza virus A (H1N1) with IC50 values of 17.9 μM, 51 μM, and 25 μM, respectively. In the first two compounds, the quinazoline-2,4-dione moiety is attached via a methylene or a butylene linker, respectively, to the 1,2,3-triazole-4-yl-β-D-ribofuranosyl fragment possessing a 5′-diphenyl phosphate substituent. In compound 17a, the uracil moiety is attached via the methylene unit to the 1,2,3-triazole-4-yl-β-D-ribofuranosyl fragment possessing a 5′-(phenyl methoxy-L-alaninyl)phosphate substituent. The remaining compounds appeared to be inactive against influenza virus A/PR/8/34/(H1N1). The results of molecular docking simulations indirectly confirmed the literature data that the inhibition of viral replication is carried out not by nucleoside analogues themselves, but by their 5′-triphosphate derivatives.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Due to high variability and rapid life cycle, influenza virus is able to develop drug resistance against direct-acting antivirals. Development of novel virus-in113039hibiting drugs is therefore important goal. Previously, we identified camphor derivative, camphecene, as an effective anti-influenza compound. In the present study, we optimize the regimen of its application to avoid high sub-toxic concentrations. The protective activity of camphecene was assessed on the model of lethal pneumonia of mice caused by influenza viruses. Camphecene was administered either once a day or four times a day, alone or in combination with Tamiflu. Mortality and viral titer in the lungs were studied. Pharmacokinetics of camphecene was studied in rabbits. We have demonstrated that camphecene, being used every 6 h at a dose of 7.5 mg/kg/day, results in antiviral effect that was statistically equal to the effect of 100 mg/kg/day once a day, that is, the same effect was achieved by 13 times lower daily dose of the drug. This effect was manifested in decrease of mortality and decrease of virus' titer in the lungs. The studies of pharmacokinetics of camphecene have demonstrated that it does not accumulate in blood plasma and that its m ultiple applications with dosage interval of 65 min are safe. In addition, the results of the study demonstrate also that camphecene possesses additive effect with Tamiflu, allowing to decrease the dose of the latter. The results suggest that due to safety and efficacy, camphecene can be further developed as potential anti-influenza remedy.

A comparative evaluation of the antiviral activity of a number of new and previously synthesized terpenophenols and their N- or O-containing derivatives against the A/Puerto Rico/8/34 (H1N1) virus strain was carried out. 2-Isobornylphenol, 1,2-dihydroxy-6-isobornyl-4-methylbenzene, 2-isobornyl-1,4-benzoquinone, and N-butyl-4-hydroxy-3,5-diisobornylbenzamide showed the highest activity.

A series of lupane-, oleanane- and dammarane-based triterpenoids with 3β-amino, A-ring azepano- and 3,4-seco-fragments has been synthesized and evaluated for antiviral activity against influenza A(H1N1) virus. It was found that azepanodipterocarpol 8 and 3β-amino-28-oxoallobetulin 11 showed antiviral activity with IC50 1.1 and 2.6 μg ml−1, and selectivity index of 19 and 10, respectively.