By using a cell-based high throughput screening campaign, a novel angelicin derivative 6a was identified to inhibit influenza A (H1N1) virus induced cytopathic effect in Madin-Darby canine kidney cell culture in low micromolar range. Detailed structure-activity relationship studies of 6a revealed that the angelicin scaffold is essential for activity in pharmacophore B, while meta-substituted phenyl/2-thiophene rings are optimal in pharmacophore A and C. The optimized lead 4-methyl-9-phenyl-8-(thiophene-2-carbonyl)-furo[2,3-h]chromen-2-one (8g, IC(50) = 70 nM) showed 64-fold enhanced activity compared to the high throughput screening (HTS) hit 6a. Also, 8g was found effective in case of influenza A (H3N2) and influenza B virus strains similar to approved anti-influenza drug zanamivir (4). Preliminary mechanistic studies suggest that these compounds act as anti-influenza agents by inhibiting ribonucleoprotein (RNP) complex associated activity and have the potential to be developed further, which could form the basis for developing additional defense against influenza pandemics.

BackgroundInfluenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.MethodsWe searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.ResultsThe 50% effective inhibitory concentration (IC50) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.ConclusionsTo the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.

H 4 )], [1] also known as the Zeise salt, in 1827, considerable numbers of olefin-coordinated transition-metal complexes have been synthesized for extensive use in organometallic and organic chemistry.[2]They also serve as useful precatalysts in the field of asymmetric catalysis. The labile olefins are rapidly and quantitatively replaced by optically active ligands that bear heteroatoms, such as nitrogen and phosphorus, which exhibit a stronger binding affinity towards metal atoms than the olefins, efficiently enabling the in situ generation of chiral catalysts.[3] However, the utilization of optically active olefin ligands as chiral modifiers had not received much attention until the independent studies of Hayashi et al. [4] and Carreira et al. [5] Their discoveries have demonstrated that chiral dienes with the appropriate geometry can form reasonably stable metal complexes due to chelation effects. These chiral diene-metal catalysts, particularly the rhodium complexes, have since been tested in catalytic asymmetric carboncarbon bond-forming reactions and were found to exhibit higher catalytic activities and enantioselectivities than phosphine ligands.[6] Since then, the design, synthesis, and investigation of chiral dienes for a wide range of asymmetric transformations has attracted much attention among synthetic organic chemists. [7, 8]

The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in MadinDarby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of capdependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5= viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.

BPR2-D2 was identified as a novel inhibitor of influenza virus. It may target viral RNPs that are responsible for viral RNA synthesis. Targeting different molecules compared with NA allows BPR2-D2 to inhibit oseltamivir-resistant viruses.

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