Synthesis and anti-influenza virus activity of 4-oxo- or thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-ones

Abstract

A target-free approach was applied to discover anti-influenza viral compounds, where influenza infected Madin-Darby canine kidney cells were treated 7500 different small organic chemicals individually and reduction of virus-induced cytopathic effect was measured. One of the hit compounds was (Z)-1-((5-fluoro-1H-indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15a) with half-maximal effective concentrations of 17.4-21.1μM against influenza A/H1N1, A/H3N2 and B viruses without any cellular toxicity at 900μM. To investigate the structure-activity relationships, two dozens of the hit analogs were synthesized. Among them, 15g, 15j, 15q, 15s, 15t and 15x had anti-influenza viral activity comparable or superior to that of the initial hit. The anti-influenza viral compounds efficiently suppressed not only viral protein level of the infected cells but also production of viral progeny in the culture supernatants in a dose-dependent manner. Based on a mode-of-action study, they did not affect virus entry or RNA replication. Instead, they suppressed viral neuraminidase activity. This study is the first to demonstrate that dihydrofuropyridinones could serve as lead compounds for the discovery of alternative influenza virus inhibitors.

Characterization and mechanisms of anti-influenza virus metabolites isolated from the Vietnamese medicinal plant Polygonum chinense

“…Here, the 4-h incubation of influenza A virus PR8 was optimized for nuclear localization of NP. The result clearly showed that both PC5 and PC7 inhibit the early steps similar to EGCG, which is a virus entry blocker [ 20 ]. As expected, PC8 did not affect NP signal intensity or its localization within 4 h after virus infection.…”

“…Cell viability was measured according to our previous report [ 20 ]. In brief, MDCK cells were seeded in 96-well plates and grown until 100% confluence.…”

“…After washing with PBS, the cells were treated with the compounds in the overlay medium for 3 days at the temperature used for virus infection. Viral plaques were counted by staining with crystal violet [ 20 ].…”

Determination of the vRNA and cRNA promoter activity by M segment-specific non-coding nucleotides of influenza A virus

“…They were then incubated with 50 µl of 2-fold diluted P0 supernatants for 1 h. After removing unabsorbed virus, the cells were incubated at 35°C for 2 or 3 days. Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay (MTT; Sigma Aldrich) as described previously[ 32 ]. For plaque assay, confluently grown MDCK cells in 48-well plates were infected with 100 µl of 10-fold serially diluted viruses at 35°C for 1 h. After washing with PBS, cells were grown in FBS-free MEM with 2 µg/ml TPCK-trypsin and 0.6% CMC (Sigma Aldrich) for 3 days[ 25 ].…”

“…Each mutation site is highlighted in a box. Synthetic promoters were subjected to the RdRp reaction as templates in the presence of ATP and [α-32 P]GTP (ppp*G). vUA(S) or cUA(s) without RdRp [(-)RdRp] was loaded as a negative control.…”