Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5.

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

Synthesis and antiviral activity of 1,2,3-triazolyl nucleoside analogues with N-acetyl-d-glucosamine residue.

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-ω-alkynyl derivatives of uracil, 6-methyluracil, thymine and 3,4,6-tri-O-acetyl-2-deoxy-2-acetamido-ß-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 µM) as the antiviral drug Rimantadine in control (IC50=77 µM) and good activity against Coxsackievirus B3 (IC50=13.9 µM) which was one and a half times higher than the activity of the antiviral drug Pleconaril in control (IC50=21.6 µM). 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.

Design of 4-Substituted Sulfonamidobenzoic Acid Derivatives Targeting Coxsackievirus B3.

A series of novel 4-substituted sulfonamidobenzoic acid derivatives was synthesized as the structural evolution of 4-(4-(1,3-dioxoisoindolin-2-yl)phenylsulfonamido)benzoic acid, which is the known inhibitor of the enterovirus life cycle. Antiviral properties of prepared compounds were evaluated in vitro using phenotypic screening and viral yield reduction assay. Their capsid binding properties were verified in thermostability assay. We identified two new hit-compounds (4 and 7a) with high activity against the coxsackievirus B3 (Nancy, CVB3) strain with potencies (IC50 values of 4.29 and 4.22 µM, respectively) which are slightly superior to the reference compound 2a (IC50 5.54 µM). Both hits changed the heat inactivation of CVB3 in vitro to higher temperatures, suggesting that they are capsid binders, as 2a is. The results obtained can serve as a basis for further development of the lead compounds for novel drug design to combat enterovirus infection.

The First 5'-Phosphorylated 1,2,3-Triazolyl Nucleoside Analogues with Uracil and Quinazoline-2,4-Dione Moieties: A Synthesis and Antiviral Evaluation.

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-ß-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-ß-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-ß-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.

Synthesis and Biological Activity of Unsymmetrical Monoterpenylhetaryl Disulfides.

New unsymmetrical monoterpenylhetaryl disulfides based on heterocyclic disulfides and monoterpene thiols were synthesized for the first time in 48-88% yields. Hydrolysis of disulfides with fragments of methyl esters of 2-mercaptonicotinic acid was carried out in 73-95% yields. The obtained compounds were evaluated for antioxidant, antibacterial, antifungal activity, cytotoxicity and mutagenicity.

Optimization of application schedule of camphecene, a novel anti-influenza compound, based on its pharmacokinetic characteristics.

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.

Synthesis and Antiviral Evaluation of Nucleoside Analogues Bearing One Pyrimidine Moiety and Two D-Ribofuranosyl Residues.

A series of 1,2,3-triazolyl nucleoside analogues in which 1,2,3-triazol-4-yl-ß-d-ribofuranosyl fragments are attached via polymethylene linkers to both nitrogen atoms of the heterocycle moiety (uracil, 6-methyluracil, thymine, quinazoline-2,4-dione, alloxazine) or to the C-5 and N-3 atoms of the 6-methyluracil moiety was synthesized. All compounds synthesized were evaluated for antiviral activity against influenza virus A/PR/8/34/(H1N1) and coxsackievirus B3. Antiviral assays revealed three compounds, 2i, 5i, 11c, which showed moderate activity against influenza virus A H1N1 with IC50 values of 57.5 µM, 24.3 µM, and 29.2 µM, respectively. In the first two nucleoside analogues, 1,2,3-triazol-4-yl-ß-d-ribofuranosyl fragments are attached via butylene linkers to N-1 and N-3 atoms of the heterocycle moiety (6-methyluracil and alloxazine, respectively). In nucleoside analogue 11c, two 1,2,3-triazol-4-yl-2',3',5'-tri-O-acetyl-ß-d-ribofuranose fragments are attached via propylene linkers to the C-5 and N-3 atoms of the 6-methyluracil moiety. Almost all synthesized 1,2,3-triazolyl nucleoside analogues showed no antiviral activity against the coxsackie B3 virus. Two exceptions are 1,2,3-triazolyl nucleoside analogs 2f and 5f, in which 1,2,3-triazol-4-yl-2',3',5'-tri-O-acetyl-ß-d-ribofuranose fragments are attached to the C-5 and N-3 atoms of the heterocycle moiety (6-methyluracil and alloxazine respectively). These compounds exhibited high antiviral potency against the coxsackie B3 virus with IC50 values of 12.4 and 11.3 µM, respectively, although both were inactive against influenza virus A H1N1. According to theoretical calculations, the antiviral activity of the 1,2,3-triazolyl nucleoside analogues 2i, 5i, and 11c against the 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). As to the antiviral activity of nucleoside analogs 2f and 5f against coxsackievirus B3, it can be explained by their interaction with the coat proteins VP1 and VP2.

Methods of Synthesis and Antiviral Activity of New 4-Alkyl-3-Nitro-1,4-Dihydroazolo[5,1-c][1,2,4]Triazin-4-ols.

An azo coupling reaction of α-nitro ketones with 5-diazoazoles was used to obtain 4-alkyl-3-nitro-1,4-dihydroazolo[5,1-с][1,2,4]triazines, which were characterized with respect to their antiviral activity against influenza and Coxsackie B3 viruses. Supplementary Information: The online version contains supplementary material available at 10.1007/s10593-021-02926-2.

New type of anti-influenza agents based on benzo[d][1,3]dithiol core.

We synthesized a series of amides with a benzo[d][1,3]dithiol core. The chemical library of compounds was tested for their cytotoxicity and inhibiting activity against influenza virus A/California/07/09 (H1N1)pdm09 in MDCK cells. For each compound, values of CC50, IC50 and selectivity index (SI) were determined. Compounds of this structure type were for the first time found to exhibit anti-influenza activity. The structure of an amide substituent in the tested compounds was demonstrated to have a significant effect on their activity against the H1N1 influenza virus and cytotoxicity. Compound 4d has a high selectivity index of about 30. 4d was shown to be most potent at early stages of viral cycle. In direct fusogenic assay it demonstrated dose-dependent activity against fusogenic activity of hemagglutinin of influenza virus. Based on molecular docking and regression analysis data, viral hemagglutinin was suggested as possible target for these new antiviral agents.

7-Imidazolyl-substituted 4'-methoxy and 3',4'-dimethoxy-containing polyfluoroflavones as promising antiviral agents.

A simple and convenient method for the synthesis of new methyl 2-(4-methoxyphenyl)- and 2-(3,4-dimethoxyphenyl)-4-oxo-4H-polyfluorochromen-3-carboxylates as analogs of natural methoxy-containing flavones is proposed. As a result of their directed modification under basic conditions, 7-imidazolyl-substituted derivatives were obtained. In aqueous-organic medium under basic conditions, 5,6,7,8-tetrafluoro-3-(methoxycarbonyl)flavones were transformed into 6,8-difluoro-5-hydroxy-7-(1H-imidazol-1-yl)-3-(methoxycarbonyl)flavones as a result of flavone-5-hydroxyflavone rearrangement, while 6,7,8-trifluorinated analogs underwent a flavone-coumarin rearrangement to give 6,8-difluoro-3-(hydroxyarylidene)-7-(1H-imidazol-1-yl)coumarins under the same conditions. Acid hydrolysis of methyl polyfluoroflavone-3-carboxylates allowed to obtain 2-aryl-4H-polyfluorochromen-4-ones. Evaluation of the antiviral activity of the synthesized compounds against influenza A (H1N1) and Coxsackie B3 viruses showed that 2-(3,4-dimethoxyphenyl)-5,6,8-trifluoro-7-(1H-imidazol-1-yl)-4H-chromene-4-one has the most promising properties.

Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents.

A chemical library was constructed based on the scaffold of camphecene (2-(E)-((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol). The modifications included introduction of mono-and bicyclic heterocyclic moieties in place of the terminal hydroxyl group of camphecene. All compounds were tested for cytotoxicity and anti-viral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells. Among 15 tested compounds 11 demonstrated a selectivity index (SI) higher than 10 and IC50 values in the micromolar range. The antiviral activity and toxicity were shown to strongly depend on the nature of the heterocyclic substituent. Compounds 2 and 14 demonstrated the highest virus-inhibiting activity with SIs of 106 and 183, and bearing pyrrolidine and piperidine moieties, correspondingly. Compound 14 was shown to interfere with viral reproduction at early stages of the viral life cycle (0-2 h post-infection). Taken together, our data suggest potential of camphecene derivatives in particular and camphor-based imine derivatives in general as effective anti-influenza compounds.

5-Chloro-2-thiophenyl-1,2,3-triazolylmethyldihydroquinolines as dual inhibitors of Mycobacterium tuberculosis and influenza virus: Synthesis and evaluation.

This study describes synthesis and evaluation of novel 5-Chloro-2-thiophenyl-1,2,3-triazolylmethyldihydroquinolines 7a-o as dual inhibitors of Mycobacterium tuberculosis and influenza virus. Huisgen's [3+2] dipolar cycloaddition of 6-(azidomethyl)-5-chloro-2-(thiophen-2-yl)-7,8-dihydroquinoline 5 with various alkynes 6a-o using sodium ascorbate and copper sulphate gave new dihydroquinoline-1,2,3-triazoles 7a-o in good to excellent yields. The new compounds were evaluated for in vitro antimycobacterial against M. tuberculosis H37Rv (Mtb) and antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1). Among the fifteen new analogs, compounds 7a (MIC: 3.12 µg/mL), 7j and 7k (MIC: 6.25 µg/mL) were identified as potent antitubercular agents. The virus-inhibiting activity of all the fifteen compounds was found to be moderate, and among them the compound 7l, bearing thiophene moiety appeared the most active with good selectivity index (IC50 = 19.5 µg/mL; SI = 15). The results presented here will help developing newer dual inhibitors of tuberculosis and influenza virus.