Synthesis and anti-influenza activities of carboxyl alkoxyalkyl esters of 4-guanidino-Neu5Ac2en (zanamivir)

Three alkoxyalkyl 2-carboxylate ester derivatives related to zanamivir were synthesized. All of the analogs of zanamivir modified at carboxylic moiety with alkoxyalkyl esters 1a-c showed higher activities than ribavirin on influenza A and B virus in the MDCK cells. Oral treatment or intraperitoneal administration of compound 1c showed significantly protective effects in mice infected with influenza A virus with low toxicities.     

Highly potent and long-acting trimeric and tetrameric inhibitors of influenza virus neuraminidase

A set of trimeric and tetrameric derivatives 6-11 of the influenza virus neuraminidase inhibitor zanamivir 1 have been synthesized by coupling a common monomeric zanamivir derivative 3 onto various multimeric carboxylic acid core groups. These discrete multimeric compounds are all significantly more antiviral than zanamivir and also show outstanding long-lasting protective activity when tested in mouse influenza infectivity experiments.     

Synthesis and anti-influenza evaluation of orally active bicyclic ether derivatives related to zanamivir

We synthesized bicyclic ether sialidase inhibitors such as tetrahydro-furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives related to zanamivir. These compounds substituted by diol at the C-3' and C-4' positions resulted in the retention of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. Compound 11a in particular showed comparable efficacy in vivo relative to that of oseltamivir phosphate.     

Synthesis and anti-influenza virus activity of 4-guanidino-7-substituted Neu5Ac2en derivatives

Substitution of 7-OH by small hydrophobic groups on zanamivir resulted in the retaining of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. These compounds were prepared by treatment of the corresponding 7-substituted sialic acids derived from 4-modified N-acetyl-D-mannosamine (ManNAc) using enzyme-catalyzed aldol condensation.     

Comparison of the anti-influenza virus activity of cyclopentane derivatives with oseltamivir and zanamivir in vivo

Cyclopentane derivatives, designated as BCX-1812, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with oseltamivir carboxylate and zanamivir for the in vivo activity in mice infected with A/Turkey/Mas/76 X A/Beijing/32/92 (H6N2) influenza virus. The compounds were tested orally and intranasally at different dose levels. BCX-1812, BCX-1827, and BCX-1923 showed more than 50% protection at 1mg/kg/day dose level on oral treatment. The intranasal treatment was 100% effective even at 0.01 mg/kg/day for all four compounds. On comparison with oseltamivir carboxylate and zanamivir, these four cyclopentane derivatives have shown equal or better efficacies. The synthesis of two new compounds, BCX-1898 and BCX-1923, is also described.     

Peptide carbocyclic derivatives as inhibitors of the viroporin function of RNA-containing viruses

New carbocyclic derivatives of amino acids, peptides, and other compounds have been synthesized, and their antiviral activity toward the influenza A/H5N1 and hepatitis C viruses has been studied in vitro. It has been shown that the aminoacyl derivatives of aminoadamantane are capable of inhibiting the replication of the highly virulent strain of the avian influenza virus (H5N1), which is resistant to aminoadamantanes amantadine and rimantadine. The effect of the configuration of the carbocyclic moiety of the dipeptide H-Pro-Trp-OH on the antiviral properties toward the hepatitis C virus has been studied. The cyclohexyloxycarbonyl derivative of the H-Pro-Trp-OH dipeptide strongly inhibited the replication of HCV in vitro. Some compounds have been found to exhibit a high virucidal activity toward influenza A/H5N1 and HCV virions.     

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.     

Heterocyclic rimantadine analogues with antiviral activity

2-(1-Adamantyl)-2-methyl-pyrrolidines 3 and 4, 2-(1-adamantyl)-2-methyl-azetidines 5 and 6, and 2-(1-adamantyl)-2-methyl-aziridines 7 and 8 were synthesized and tested for their antiviral activity against influenza A. Parent molecules 3, 5, and 7 contain the alpha-methyl-1-adamantan-methanamine 2 pharmacophoric moiety (rimantadine). The ring size effect on anti-influenza A activity was investigated. Pyrrolidine 3 was the most potent anti-influenza virus A compound, 9-fold more potent than rimantadine 2, 27-fold more potent than amantadine 1, and 22-fold more potent than ribavirin. Azetidines 5 and 6 were both markedly active against influenza A H2N2 virus, 10- to 20-fold more potent than amantadine. Aziridine 7 was almost devoid of any activity against H2N2 virus but exhibited borderline activity against H3N2 influenza A strain. Thus, it appears that changing the five-, to four- to a three-membered ring results in a drop of activity against influenza A virus.     

Effect of d-Limonene and related compounds on bile flow and biliary lipid composition in rats and dogs

d-Limonene enhanced bile flow in rats and dogs with a dose response correlation. The choleretic activity was much higher in the metabolites of d-limonene such as p-mentha-1,8-dien-10-ol, p-menth-1-ene-8,9-diol and p-mentha-1,8-dien-6-ol than d-limonene, and this suggested that the choleretic activity of d-limonene was attributable at least in part to its metabolites.The choleretic activities of esters of p-menth-1-ene-8,9-diol with acetic acid, propionic acid, stearic acid, palmitic acid, linoleic acid, benzoic acid, salicylic acid, α-naphthylacetic acid and nicotinic acid were also investigated in rats. Among these compounds, acetate, propionate and nicotinate possessed considerable, but lesser activities than the original diol. In dogs, however, the choleretic activity of p-menth-l-ene-8,9-diol acetate and propionate was much higher than that of original diol, suggesting that the choleretic activity of these esters is attributable to the esters themselves.d-Limonene decreased the ratio of biliary bile salts and phospholipids to cholesterol, whereas p-menth-l-ene-8,9-diol increased it.     

A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.     

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.     

Studies on antiviral glycosides. Synthesis and biological evaluation of various phenyl glycosides.

A variety of analogues and derivatives of phenyl glycosides were synthesized for examination of their biological activities and of the relationship between structure and antiviral activity. For antiviral activity, a 6-deoxy-6-halogeno-D-glucose residue was most suitable for the carbohydrate moiety and p-alkylphenyl groups for the aglycone moiety. Based on these results, p-(sec-butyl)phenyl 6-chloro-6-deoxy-beta-D-glucopyranoside and p-(sec-butyl)phenyl 6-deoxy-6-iodo-beta-D-glucopyranoside were prepared, and the former compound was found to be the most potent antiviral substance, in this series, against influenza and Herpes simplex virus. The anomeric configuration of phenyl glycosides did not contribute to the antiviral activity.     

Dihydromyricetin is a new inhibitor of influenza polymerase PB2 subunit and influenza-induced inflammation

Development of new and effective anti-influenza drugs is critical for the treatment of influenza virus infection. The polymerase basic 2 (PB2) subunit as a core subunit of influenza A virus RNA polymerase complex is considered to be an attractive drug target for anti-influenza drug discovery. Dihydromyricetin, as a natural flavonoid, has a wide range of biological activities, but its anti-influenza A virus activity is ambiguous. Here, we found dihydromyricetin could inhibit the replication of a variety of influenza A virus strains. Mechanism studies demonstrated that dihydromyricetin reduced viral polymerase activity via selective inhibition of viral PB2 subunit, and decreased relative amounts of viral mRNA and genomic RNA during influenza A virus infection. The binding affinity and molecular docking analyses revealed that dihydromyricetin interacted with the PB2 cap-binding pocket, functioned as a cap-binding competitor. Interestingly, dihydromyricetin also reduced cellular immune injury by inhibiting TLR3 signaling pathway. Additionally, combination treatment of dihydromyricetin with zanamivir exerted a synergistic anti-influenza effect. Altogether, our experiments reveal the antiviral and anti-inflammatory activities of dihydromyricetin in vitro against influenza virus infection, which provides a new insight into the development of novel anti-influenza drugs.     

Syntheses of triazole-modified zanamivir analogues via click chemistry and anti-AIV activities

Sixteen novel 4-triazole-modified zanamivir (1) analogues were synthesized using the click reactions, and their inhibitory activities against avian influenza virus (AIV, H5N1) were determined. Compound 3b exerts promising inhibitory activity with EC(50) of 6.4 microM, which is very close to that of zanamivir (EC(50) = 2.8 microM). Molecular modeling provided the information about the binding model between inhibitors and neuraminidase, which are in good agreement with inhibitory activities.     

Mutagenicity of non-K-region diols and diol-epoxides of benz(a)anthracene and benzo(a)pyrene in S. typhimurium TA 100

When incubated with a 9,000 x g rat-liver supernatant, benzo(a)pyrene 7,8-diol and benz(a)anthracene 8,9-diol were more active than the parent hydrocarbons in inducing his⁺ revertant colonies of S. typhimurium TA 100. Benzo(a) pyrene 9,10-diol was less active than benzo(a)pyrene; the K-region diols, benz(a)anthracene 5,6-diol and benzo(a)pyrene 4,5-diol, were inactive. None of the diols was active when the cofactors for the microsomal mono-oxygenase were omitted. The diol-epoxides benzo(a)pyrene 7,8-diol 9,10-oxide, benz(a)anthracene 8,9-diol 10,11-oxide and 7-methylbenz(a)anthracene 8,9-diol 10,11-oxide and the K-region epoxides, benzo(a)pyrene 4,5-oxide and benz(a)anthracene 5,6-oxide, were mutagenic without further metabolism.     

Synthesis and anti-influenza virus activity of dihydrofuran-fused perhydrophenanthrenes with a benzyloxy-type side-chain

As one of our ongoing research project concerning development of a novel anti-influenza virus agent, dihydrofuran-fused perhydrophenanthrenes were derivatized by means of Williamson ether synthesis and Suzuki–Miyaura cross coupling reactions. Newly synthesized compounds were subjected to evaluation of anti-influenza virus activity using influenza A/Aichi/2/68 (H3N2 subtype) virus strain by a plaque titration method. These investigations revealed that incorporation of benzyl-type ether substituents was effective for exerting the inhibition activity of influenza virus proliferation.     

Current status for influenza control

Influenza viruses are responsible for respiratory illness with significant morbidity and mortality. To curb the disease, two-pronged attack on the virus, therapeutic and prophylactic, is being actively pursued. The therapeutic use of existing anti-influenza drugs, such as amantadine and rimantadine, is limited by their significant adverse side effect, emergence of resistant viral strains, and lack of activity against influenza B virus. A new class of antiviral agents designed to inhibit influenza neuraminidase are currently under active development for use in the prophylaxis and treatment of influenza A and B virus infections. Two of these compounds, zanamivir (GG167) and GS4104 have reached clinical trials. Limitations in the effectiveness and application of inactivated vaccines have stimulated development of alternative approaches to influenza immunization. One such approach is a live, intranasally administered vaccine, attenuated by cold-adaptation of a master strain with subsequent genetic reassortment with circulating wild-type strains. Recently developed reverse-genetics techniques have made it possible to use RNA viruses as vector. Besides DNA viral vectors, live influenza virus vectors may emerge as a useful alternative for the vaccination against different pathogens.     

Zanamivir: from drug design to the clinic.

The development of the neuraminidase inhibitors has revolutionized the management options for influenza. Zanamivir was the first such inhibitor to be approved for the treatment of influenza in humans. It is delivered by inhalation to the respiratory tract, which is the site of viral replication, in order to ensure immediate antiviral activity. Early treatment with zanamivir in clinical trials rapidly reduced the severity and duration of influenza symptoms and associated complications. Furthermore, chemoprophylaxis with zanamivir was shown to be effective in the prevention of influenza illness. To date, there is no evidence for the emergence of clinically significant zanamivir-resistant isolates. In conclusion, zanamivir offers a useful complementary strategy to vaccination in the effective management of influenza.     

Bacterial Neuraminidase Rescues Influenza Virus Replication from Inhibition by a Neuraminidase Inhibitor

Influenza virus neuraminidase (NA) cleaves terminal sialic acid residues on oligosaccharide chains that are receptors for virus binding, thus playing an important role in the release of virions from infected cells to promote the spread of cell-to-cell infection. In addition, NA plays a role at the initial stage of viral infection in the respiratory tract by degrading hemagglutination inhibitors in body fluid which competitively inhibit receptor binding of the virus. Current first line anti-influenza drugs are viral NA-specific inhibitors, which do not inhibit bacterial neuraminidases. Since neuraminidase producing bacteria have been isolated from oral and upper respiratory commensal bacterial flora, we posited that bacterial neuraminidases could decrease the antiviral effectiveness of NA inhibitor drugs in respiratory organs when viral NA is inhibited. Using in vitro models of infection, we aimed to clarify the effects of bacterial neuraminidases on influenza virus infection in the presence of the NA inhibitor drug zanamivir. We found that zanamivir reduced progeny virus yield to less than 2% of that in its absence, however the yield was restored almost entirely by the exogenous addition of bacterial neuraminidase from Streptococcus pneumoniae. Furthermore, cell-to-cell infection was severely inhibited by zanamivir but restored by the addition of bacterial neuraminidase. Next we examined the effects of bacterial neuraminidase on hemagglutination inhibition and infectivity neutralization activities of human saliva in the presence of zanamivir. We found that the drug enhanced both inhibitory activities of saliva, while the addition of bacterial neuraminidase diminished this enhancement. Altogether, our results showed that bacterial neuraminidases functioned as the predominant NA when viral NA was inhibited to promote the spread of infection and to inactivate the neutralization activity of saliva. We propose that neuraminidase from bacterial flora in patients may reduce the efficacy of NA inhibitor drugs during influenza virus infection. (295 words).