Author's personal copy 4530 W.-H. Song ef al / Bioorg Med. Chem. Lett. 23(2013)4528-4531 Anti-HCV activities for the 24 synthesized compounds Compound Inhibition ratioe- 50 HM(%) 1234 xNNNNNNNNNNNNNcccccccc 28.70 62 20 12345 64 Furan-2-yl 14.5 34 3-(Benzyloxy )phenyl 3-(4-Fluorobenzyloxy )phenyl 29 azyloxy )phenyl C (4-Fluorobenzyl)furan-2-yl RG7128 75.3%@2pM The valuated in an authentic HCV infection/replication system by measured the EgFP autofluorescence in Huh-7 cell lines. The values represent The best three which were further selected to determine the ECso values. Table 2 25 CCso(uM) sid >500 >77 Cv assay was evaluated in an authentic HCv infection/ replication system by measured the EGFP autofluorescence in Huh-7 cell lines. The ECso value of each compound was the concentration required to inhibit HCV RNA replication by 50% and was estimated by lir ition of 5 conce ons. The values represent average of triplicate results. 005 The CCso value of each compound means concentration required to reduce cell proliferation by50‰. Selectivity index(Si): ratio of CCso to eC uM 1 HM 5 AM 10 AM RG7128 Compound 30 the reductionof the viral protein and mrNa levels we ompound 30 in ndent manner. RG7128(2 HM) served as positive control and relative by western blot and Real-time PCR. This study disc ne levels were calculated as a ratio of the hcv genome levels to gADPh lead compound to design more potent anti-HCV structural modification of compound 30 as an anti-HCV candidate RG7128 is currently in progress. Mock Acknowledge S5A This work was supported by grants from the National Natural lin Science Foundation of China(No. 20902013 and No 30973641) National Basic Research Program of China(No. 2009CB522504) ompound 30 in a dose-dependent manner. RG719 pression was inhibited by Shanghai Committee of Science and Technology of China(Grant Figure 3. Western blotting showed that HC No 13ZR1403900), China Postdoctoral Science Foundation(No. control and tubulin was loading controL 2013M531126), and"Zhuo Xue "Program of Fudan UniversiAuthor's personal copy the reductionof the viral protein and mRNA levels were also tested by western blot and Real-time PCR. This study discovered a new lead compound to design more potent anti-HCV agents. Further structural modification of compound 30 as an anti-HCV candidate is currently in progress. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 20902013 and No. 30973641), National Basic Research Program of China (No. 2009CB522504), Shanghai Committee of Science and Technology of China (Grant No.13ZR1403900), China Postdoctoral Science Foundation (No. 2013M531126), and "Zhuo Xue" Program of Fudan University. Table 1 Anti-HCV activities for the 24 synthesized compounds Ar O O N N X H N Compound Ar X Inhibition ratio@a 50 lM (%) 10 Phenyl N 1.3 11 Furan-2-yl N 0.9 12 4-Nitrophenyl N 14.74 13 4-Methoxyphenyl N 28.70 14 2-(Benzyloxy)phenyl N 67.6c 15 3-(Benzyloxy)phenyl N 11.1 16 4-(Benzyloxy)phenyl N 54.9 17 2-(4-Fluorobenzyloxy)phenyl N 6.8 18 3-(4-Fluorobenzyloxy)phenyl N 62.7 19 4-(4-Fluorobenzyloxy)phenyl N 10.7 20 2-(4-Chlorobenzyloxy)phenyl N 36.2 21 3-(4-Chlorobenzyloxy)phenyl N 43.6 22 4-(4-Chlorobenzyloxy)phenyl N 64.9 23 Phenyl C 24.8 24 Furan-2-yl C 14.5 25 2-(Benzyloxy)phenyl C 34.4 26 3-(Benzyloxy)phenyl C 48.2 27 4-(Benzyloxy)phenyl C 12.1 28 2-(4-Fluorobenzyloxy)phenyl C 55.0 29 3-(4-Fluorobenzyloxy)phenyl C 43.4 30 4-(4-Fluorobenzyloxy)phenyl C 69.7c 31 2-(4-Chlorobenzyloxy)phenyl C 53.8 32 3-(4-Chlorobenzyloxy)phenyl C 29.2 33 4-(4-Chlorobenzyloxy)phenyl C 70.2c 5 (5-Chloroindol)-3-yl N 22.4 6 (4-Fluorobenzyl)furan-2-yl C 24.3 RG7128b 75.3% @ 2 lM a The anti-HCV assay was evaluated in an authentic HCV infection/replication system by measured the EGFP autofluorescence in Huh-7 cell lines. The values represent a means of triplicate results. b RG7128 was used as a reference positive control. c The best three compounds which were further selected to determine the EC50 values. Table 2 Anti-HCV antivities (EC50) and cytotoxicities (CC50) for selected 3 compounds Compound EC50a,b (lM) CC50c (lM) SId 14 9.2 >500 >54 30 3.9 >500 >128 33 6.5 >500 >77 a The anti-HCV assay was evaluated in an authentic HCV infection/replication system by measured the EGFP autofluorescence in Huh-7 cell lines. b The EC50 value of each compound was the concentration required to inhibit HCV RNA replication by 50% and was estimated by linear interpolation from inhi￾bition of 5 concentrations. The values represent average of triplicate results. c The CC50 value of each compound means concentration required to reduce cell proliferation by 50%. d Selectivity index(SI): ratio of CC50 to EC50. Figure 3. Western blotting showed that HCV protein expression was inhibited by compound 30 in a dose-dependent manner. RG7128 (2 lM) served as positive control and tubulin was loading control. Figure 4. QRT-PCR showed that HCV replication was inhibited by compound 30 in a dose-dependent manner. RG7128 (2 lM) served as positive control and relative HCV genome levels were calculated as a ratio of the HCV genome levels to GADPH mRNA levels. 4530 W.-H. Song et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4528–4531