Antiviral Evaluation Research Articles

Basimarols A, B, and C, Highly Oxygenated Pimarane Diterpenoids from Basilicum polystachyon

The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3-5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3-5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.

Design, synthesis and antiviral evaluation of novel acyclic phosphonate nucleotide analogs with triazolo[4,5-b]pyridine, imidazo[4,5-b]pyridine and imidazo[4,5-b]pyridin-2(3H)-one systems

A new series of phosphonylated triazolo[4,5-b]pyridine (1-deaza-8-azapurine), imidazo[4,5-b]pyridine (1-deazapurine) and imidazo[4,5-b]pyridin-2(3H)-one (1-deazapurin-8-one) were synthesized from 2-chloro-3-nitropyridine and selected diethyl ɷ-aminoalkylphosphonates followed by reduction of the nitro group and cyclization. In the final step O,O-diethylphosphonates were transformed into the corresponding phosphonic acids. All synthesized compounds were evaluated in vitro for inhibitory activity against a broad variety of DNA and RNA viruses and their cytotoxic potencies were also established. Compound 12f showed marginal activity against cytomegalovirus Davis strain (EC50 = 76.47 μM) in human embryonic lung (HEL) cells while compounds 10g (EC50 = 52.53 μM) and 12l (EC50 = 61.70 μM) were minimally active against the varicella-zoster virus Oka strain in HEL cells. Compounds under investigation were not cytotoxic at the maximum concentration evaluated (100 µM).

Pathogenicity of fowl adenovirus serotype 4 (FAdV-4) in chickens

Hepatitis-hydropericardium syndrome (HHS) is an acute infectious disease caused by fowl adenovirus serotype 4 (FAdV-4), which mainly infects broilers aged 3–5 weeks. In March 2018, a pathogenic disease, which was characterized by symptoms similar to HHS, broke out in 10-day-old broiler flocks in Shandong province. In this study, a strain of FAdV-4 (SDSG) was isolated from naturally infected broilers. To assess its pathogenicity, 10-day-old and 20-day-old specific pathogen-free (SPF) chickens were inoculated separately with the FAdV-4 virus fluid via oral and intramuscular injection routes.The results show that typical hydropericardium and hepatitis were observed in experimental chickens. The titer levels of the virus antibody and the levels of inflammatory cytokines were upregulated, which may be caused by the infection and innate immune response. The detection of viral load showed that the presence of virus was detected in multiple organs, in which the liver contained the highest concentration of viral DNA, and the virus content in the intramuscular injection group was higher than that of the oral injection group.In summary, these findings increase our understanding of the pathogenicity of FAdV-4 (SDSG) in chickens. The established model will be valuable for anti-viral drug testing and vaccine evaluation, which can prevent and reduce the spread of HHS in the poultry industry.

Synthesis of D-like and L-like 5′-fluoro-5′-deoxy-1′,6′-isoneplanocins and their antiviral activity including Ebola

In exploring the 1′,6′-isoneplanocin structural framework for antiviral compound development, both the D-like and L-like 5′-fluoro-5′-deoxy analogs have been synthesized from readily available cyclopentenyl iodides and adenine in five steps. Antiviral evaluation found D-like 5′-fluoro-5′-deoxy-1′,6′-isoneplanocin to display less broad-spectrum antiviral effects than the parent D-like including weaker activity against Ebola and norovirus. The L-like enantiomer was devoid of any activity, contrary to the parent L-like compound.

Botryosphaeran and sulfonated derivatives as novel antiviral agents for herpes simplex and dengue fever

Sulfated polysaccharides are known to display activity against enveloped viruses, such as herpes and dengue. The aim of this work was to assess the antiviral activity of botryosphaeran, a fungal exocellular (1 → 3)(1 → 6)-β-d-glucan devoid of sulfate groups, and its chemically sulfonated derivatives, against herpes simplex virus (HSV), dengue virus (DENV) and poliovirus (PV). The natural parent polysaccharide inhibited acyclovir-sensitive HSV (HSV-KOS) infection in Vero cells (IC50 of 39.3 μg mL−1), while the IC50 against acyclovir-resistant HSV (HSV-AR) was 47.5 μg mL−1. Botryosphaeran was derivatized by sulfonylation with chlorosulfonic acid to prepare two sulfonated derivatives, S1 and S2, with degrees of substitution (DS) of 0.4 and 1.1, respectively. Antiviral evaluation of S1 and S2 gave the IC50 of 3.0 and 2.4 μg mL−1 against HSV-KOS, and 7.3 and 2.7 μg mL−1 against HSV-AR, respectively. This study demonstrated for the first time that native botryosphaeran inhibited HSV infection, albeit moderately, while its sulfonated derivatives developed high activity against viral infection. DENV inhibition was weak for botryosphaeran, but remarkably stronger for S1 and S2. All compounds were inactive against PV, as it lacked a viral envelope. The presence of sulfate groups and the DS were confirmed to be important features for antiviral activity.

Synthesis, Spectral Characterization, Docking Studies and Antiviral Activity of Phosphorylated Derivatives of Lopinavir Intermediate

AbstractLopinavir (LPV) intermediate, a core unit of the HIV‐protease inhibitor is phosphorylated and screened for their binding efficacy with the Hemagglutinin‐Neuraminidase (HN) protein of Newcastle disease virus (NDV) and of VP7 protein of Bluetongue virus (BTV). The docking simulations were encouraged for their synthesis and screened their antiviral activity against NDV in the Chicken Embryonated eggs. The compounds which have exhibited higher binding energy were selected for antiviral evaluation on target pathogens such as NDV and BTV using in in ovo and in vitro methods. The results revealed that the compounds, 5a, 5b, 5i and 5j showed good activity against NDV and 5a, 5b, 5g and 5j exhibited noble antiviral activity against BTV in both in ovo and in vitro than the remaining title compounds.

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Antiviral study on Punica granatum L., Momordica charantia L., Andrographis paniculata Nees, and Melia azedarach L., to Human Herpes Virus-3

IntroductionHuman Herpes Virus-3 (Varicella Zoster Virus) causes Chickenpox in childhood and reactivates after decades of being latent to cause Herpes Zoster in adults. The aim of this study was to evaluate the in vitro antiviral potency on the leaves of Punica granatum L., Momordica charantia L., Andrographis paniculata Nees. and Melia azedarach L., against the Human Herpes Virus-3 isolated from Chickenpox and Zoster in comparison with acyclovir. MethodsAqueous, ethanolic and aqueous ethanolic extracts were prepared from the chosen plant leaves by lyophilization process and subjected to in vitro cytotoxicity assay in HEp-2 cells followed by the in vitro antiviral evaluation against the clinical isolates of HHV-3 using post incubation assay. The structure of leaf chemicals were retrieved from protein data bank and in silico drug analysis was carried out through discovery studio targeting the protease of HHV-3.The drug likeliness and the ADMETSAR properties of the screened active phytochemicals were calculated. ResultsAqueous extract from the leaves of Punica granatum L., exhibited potential antiviral activity against the HHV-3. The in silico docking results found that the phytochemicals of Punica granatum L., interacted on the active site of the HHV-3 protease. ConclusionAqueous extract from the leaves of Punica granatum L. was superior in exhibiting its antiviral efficacy to HHV-3 whose in vitro activity was comparable with acyclovir. As the leaf phytochemicals interacted with the HHV-3 protease, the antiviral activity of the Punica granatum L., leaves may interfere with the capsid assembly of the HHV-3.

Design, Synthesis, Antiviral Evaluation, and SAR Studies of New 1-(Phenylsulfonyl)-1H-Pyrazol-4-yl-Methylaniline Derivatives.

A series of N-((3-phenyl-1-(phenylsulfonyl)-1H-pyrazol-4-yl)methyl)anilines 7a-p and 8a-l, structurally related to previously synthesized and tested (N-(1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines (1a-v), were designed and synthesized. The new derivatives were evaluated in cell-based assays for their cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses of public health significance. Generally, the tested compounds did not display cytotoxicity toward the cell lines used. The majority of derivatives 7a-p were able to interfered with YFV and RSV replication in the micromolar range showing a marked improvement in potency and selectivity with respect to the reference inhibitors 6-azauridine and ribavirin, respectively. The introduction of a p-methoxy substituent on the phenylsulfonyl group (compounds 8a-l) completely abolished the anti-RSV activity and reduced or eliminated the potency against YFV. On the contrary, several p-methoxy analogs were able to interfere with BVDV replication with a comparable (8b, 8c, 8g, and 8k) or better (8a and 8f) potency than the reference inhibitor, ribavirin. Compound 7e, selected for time of addition experiments on BHK-21 cell cultures infected with YFV, achieved the highest reduction of virus titer when added 2 h post infection and maintained up to 4 h post infection.

Cell line-dependent activation and antiviral activity of T-1105, the non-fluorinated analogue of T-705 (favipiravir)

The antiviral drug T-705 (favipiravir) and its non-fluorinated analogue T-1105 inhibit the polymerases of RNA viruses after being converted to their ribonucleoside triphosphate (RTP) metabolite. We here compared the activation efficiency of T-705 and T-1105 in four cell lines that are commonly used for their antiviral evaluation. In MDCK cells, the levels of T-705-RTP were markedly lower than those of T-1105-RTP, while the opposite was seen in A549, Vero and HEK293T cells. In the latter three cell lines, T-1105 activation was hindered by inefficient conversion of the ribonucleoside monophosphate to the ribonucleoside diphosphate en route to forming the active triphosphate. Accordingly, T-1105 had better anti-RNA virus activity in MDCK cells, while T-705 was more potent in the other three cell lines. Additionally, we identified a fourth metabolite, the NAD analogue of T-705/T-1105, and showed that it can be formed by nicotinamide mononucleotide adenylyltransferase.

3-Trifluoromethylpyrazolones derived nucleosides: Synthesis and antiviral evaluation

Dengue (DENV) viral infection is a global public health problem that infrequently develops life threatening diseases such as dengue hemorrhagic fever (DFS) and dengue shock syndrome (DSS). Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human corona virus with 38% fatality rate of infected patients. A series of 4-arylhydrazono-5-trifluoromethyl-pyrazolones, their ribofuranosyl, and 5′-deoxyribofuranosyl nucleosides were synthesized, geometry optimized using Density functional theory (DFT), and evaluated for their antiviral activity. 2-Nitrophenylhydrazonopyra-zolone derivative 5 showed significant activity against MERS-CoV (EC50 = 4.6 μM). The nucleoside analog 8 showed moderate activity against DENV-2 (EC50 = 10 μM), while the activity was abolished with the corresponding 5′-deoxyribonucleoside analogs. The identified hits in this study set this category of compounds for further future optimizations.

Micropatterned Viral Membrane Clusters for Antiviral Drug Evaluation.

The function of biological nanoparticles, such as membrane-enveloped viral particles, is often enhanced when the particles form higher-order supramolecular assemblies. While there is intense interest in developing biomimetic platforms that recapitulate these collective properties, existing platforms are limited to mimicking individual virus particles. Here, we present a micropatterning strategy to print linker molecules selectively onto bioinert surfaces, thereby enabling controlled tethering of biomimetic viral particle clusters across defined geometric patterns. By controlling the linker concentration, it is possible to tune the density of tethered particles within clusters while enhancing the signal intensity of encapsulated fluorescent markers. Time-resolved tracking of pore formation and membrane lysis revealed that an antiviral peptide can disturb clusters of the membrane-enclosed particles akin to the targeting of individual viral particles. This platform is broadly useful for evaluating the performance of membrane-active antiviral drug candidates, whereas the micropatterning strategy can be applied to a wide range of biological nanoparticles and other macromolecular entities.

Synthesis and Antiviral Evaluation of 3'-Fluoro-4'-modified-5'-norcarbocyclic Nucleoside Phosphonates.

The synthesis and anti-HIV evaluation of hitherto unknown 3'-fluoro-5'-norcarbocyclic nucleoside phosphonates bearing adenine with modifications at the 4' position (ethynyl, vinyl, ethyl, hydroxymethyl) is described. One of the synthesized compounds was found to be an inhibitor of HIV-1 replication, but with moderate efficiency relative to (R)-9-(2-phosphonylmethoxypropyl)adenine ((R)-PMPA, tenofovir), with no concomitant cytotoxicity.

Cytotoxicity and antiviral activity evaluation of Cymbopogon spp hydroethanolic extracts

Cymbopogon citratus and C. nardus are noteworthy among the several existing plant species displaying medicinal properties, due to the potential pharmacological activity of these species, including antiviral, antibacterial, antifungal and anti-trypanosomal activities. The objective of this study was to carry out in vitro toxicity tests of plant extracts from both species and analyze potential antiviral activity against Human mastadenovirus serotype 5 (HAdV-5). Two cell lines (A549 and VERO) were used and mitochondrial and lysosomal viability were determined by the MTT and neutral red assay, respectively, after two exposure times (24 hours and six days). The aim of these assays was to counteract the behavior of the extracts against the different cell lines and determine their non-toxic concentration range, in order to evaluate possible antiviral activity against HAdV-5. Plaque reduction and inhibition index of viral titer assays were performed using the maximum non-cytotoxic concentrations (MNCC) of each extract. The results indicate MNCC at 625 μg/mL for all extracts, except for Cymbopogon nardus obtained with 80% ethanol (CN80), which showed toxicity at concentrations higher than 312.5 μg/mL. CN80 was the only extract that displayed potential activity against HAdV-5, at a concentration of 75 μg/mL, becoming a candidate for extract fraction purification and/or the isolation of substances related to the observed antiviral activity.

Synthesis, Antiviral Evaluation and Molecular Docking Studies of Azo Compounds

Synthesis, Antiviral Evaluation and Molecular Docking Studies of Azo Compounds

MECHANISM OF ANTIVIRAL ACTION AND EVALUATION OF THE EFFICACY OF THE NEW PREPARATION FORTEPREN<sup>®</sup> IN THE COMPLEX THERAPY OF CHRONIC RECURRENT HERPESVIRUS INFECTION OF GENITAL LOCALIZATION

Aim. The study of the mechanism of antiviral action and evaluation of the clinical efficacy, safety and tolerability of therapy with Fortepren® in patients with chronic recurrent herpesvirus infection of genital localization (CRHVI). Materials and methods. Clinical studies were carried out of a drug Fortepren® (0.4% sodium polyprenyl phosphate solution), which was administered to patients who underwent a basic therapeutic course of the drug Acyclovir-Acry® to relieve the acute phase of the disease. The study was performed on 80 male and female patients selected during the screening with a confirmed diagnosis of CRHVI. Two groups were formed. Patients of group 1 (experimental) were intramuscularly injected with Fortepren® at a dose of 2 ml (8 mg) three times at intervals of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days following the 10-day basic course of treatment of the acute phase of diseases with the use of the acyclovir tablets of 400 mg - 13 ± 2, 34 ± 2 and 55 ± 2 days from the beginning of the study. Patients of the 2nd group (control) were intramuscularly injected with placebo solution at a volume of 2 ml instead of Fortepren®. To evaluate Fortepren® efficacy, the following criteria were used: increase in the duration of the inter-recessive period, a decrease in the frequency of relapses over the entire observation period; decrease in the severity of relapses, estimated in points, changes in immunological parameters according to the dynamics of changes in the production of the main cytokines. Results. In patients treated with Fortepren®, the inter-recurrence period for the entire study period increased statistically from 29.36 ± 2.16 to 42.98 ± 3.29 days, while in the control group this indicator have not changed. Accordingly, in patients treated with Fortepren®, a statistically significant reduction in the incidence of recurrence of CRHVI from 3.03 ± 0.02 before treatment to 1.94 ± 0.19 was observed during treatment in the absence of a decrease in the frequency of relapses in the control. Evaluation of the severity of CRHVI relapses in patients treated with Fortepren® indicates the efficacy of this protocol. The sum of the scores of the mean values of CRHVI symptoms signs was statistically significantly decreased in the group 1 from 7.36 ± 0.35 points at the 1 st visit before the start of treatment to 4.75 ± 0.35 points during the treatment. No changes were seen in the control group. The level of leukocyte virus-induced interferon (LVI-IFN) in the patients of the group 1 increased from 36% to 64% in the end of the clinical trial compared to the control group, in which the increase in LVI-IFN titers was not observed. To further justify the possibility of increasing the immune response of cells, establishing possible mechanisms that determine the efficacy of treatment for CRHVI with Fortepren®, evaluation of the production of IFNz, IFNy, IL-10, IL-12p40, IL-12p70, IL-15, IL-2, IL-4, MIF-Fz, TNFа was made. In the of the study levels of all these cytokines was increased in patients treated with Fortepren® compared with the control group. Conclusion. The efficacy of using Fortepren® in a dose of 2 ml (8 mg) with intramuscular administration to patients with chronic recurrent herpesviral infection of genital localization at the stage of remission three times with an interval of 21 days by 3 ± 2, 24 ± 2 and 45 ± 2 days after the end of 10 day basic course of treatment of the acute phase of the disease with the use of the drug acyclovir.

Ebola viral dynamics in nonhuman primates provides insights into virus immuno-pathogenesis and antiviral strategies

Despite several clinical trials implemented, no antiviral drug could demonstrate efficacy against Ebola virus. In non-human primates, early initiation of polymerase inhibitors favipiravir and remdesivir improves survival, but whether they could be effective in patients is unknown. Here we analyze the impact of antiviral therapy by using a mathematical model that integrates virological and immunological data of 44 cynomolgus macaques, left untreated or treated with favipiravir. We estimate that favipiravir has a ~50% efficacy in blocking viral production, which results in reducing virus growth and cytokine storm while IFNα reduces cell susceptibility to infection. Simulating the effect of delayed initiations of treatment, our model predicts survival rates of 60% for favipiravir and 100% for remdesivir when treatment is initiated within 3 and 4 days post infection, respectively. These results improve the understanding of Ebola immuno-pathogenesis and can help optimize antiviral evaluation in future outbreaks.

Synthesis and Antiviral Evaluation of Carbocyclic Nucleoside Analogs of Nucleoside Reverse Transcriptase Translocation Inhibitor MK-8591 (4'-Ethynyl-2-fluoro-2'-deoxyadenosine).

MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine) is a novel nucleoside analog that displays a differentiated mechanism of action as a nucleoside reverse transcriptase translocation inhibitor (NRTTI) compared to approved NRTIs. Herein, we describe our recent efforts to explore the impact of structural changes to the properties of MK-8591 through the synthesis and antiviral evaluation of carbocyclic derivatives. Synthesized analogs were evaluated for their antiviral activity, and the corresponding triphosphates were synthesized and evaluated in a biochemical assay. 4'-Ethynyl-G derivative (±)-29 displayed a promising IC50 of 33 nM in a hPBMC cell-based antiviral assay, and its triphosphate (TP), (±)-29-TP, displayed an IC50 of 324 nM in a biochemical RT-polymerase assay. Improved TP anabolite delivery resulting in improved in vitro potency was achieved by preparing the corresponding phosphoramidate prodrug of single enantiomer 29b, with 6-ethoxy G derivative 34b displaying a significantly improved IC50 of 3.0 nM, paving the way for new directions for this novel class of nucleoside analogs.

Synthesis and Utility of α,β‐Unsaturated Ketone Bearing Naphthalene and Benzofuran Rings in the Synthesis of Some N‐heterocycles with Their Antiviral and Antitumor Activity Evaluation

A chalcone bearing naphthalene–benzofuran rings was synthesized. The behavior of this chalcone towards some nitrogen and carbon nucleophiles with formation of pyrazole, isooxazole, thiazole, pyrimidine, pyran, and pyridine rings was studied. Also, pyridine acetohydrazide was synthesized and utilized as a key starting material in the synthesis of 2,4‐dioxothiazolidine and 2,4‐dioxoquinazolin derivatives. The structures of all the products obtained were illustrated from their analytical and spectral data. Some new compounds were selected and evaluated as antiviral agents against hepatitis A virus. In addition, the antitumor activities of certain selected new compounds were screened, in vitro, against HepG2 and MCF‐7 cell lines.

Design, synthesis, and antiviral evaluation of novel hydrazone-substituted thiophene[3,2-d]pyrimidine derivatives as potent human immunodeficiency virus-1 inhibitors.

In the previous studies of our laboratory, the thiophene[3,2-d]pyrimidine was identified as a promising scaffold for seeking highly potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, we designed, synthesized, and biologically evaluated a series of thiophene[3,2-d]pyrimidine derivatives with changed linker between the thiophenepyrimidine core and the right wing. Some of the synthesized compounds exhibited excellent HIV-1 inhibitory potency with low (double-digit) nanomolar 50% effective concentration (EC50 ) values. Among them, compound 13a exhibited the most potent anti-HIV-1 activity (EC50 =21.2nM), which was 10-fold greater than that of NVP (EC50 =281nM). Moreover, 13a showed much lower cytotoxicity (CC50 =183μM) and higher selection index (SI=8,632) than NVP, ETV, and AZT. Besides, some physicochemical properties and water solubility were calculated or measured. The preliminary structure-activity relationships and molecular simulation studies of these compounds were also discussed comprehensively to provide valuable direction for further design and optimization.