1 Drugs against HCV infection are facing several drawbacks such as undesirable side effects, 2 emerging of HCV resistant strains, and high cost of the entire course of treatment. Thus, 3 new active and cost-effective compounds are required to develop effective drugs to combat 4 HCV infection. This study was designed to test the antiviral activity of quinazoline derivatives 5 against HCV infection using HCV protease assay (HCV NS3-4Apro) and cell-based HCV 6 replicon assay. The results showed that some of quinazoline derivatives inhibited HCV NS3-7 4Apro with IC 50 ranged from 42 µM of compound 4 to 150 µM of compound 2. In this study, 8 compound 4 was considered as the best compound lead to developing potent anti-HCV 9 NS3-4Apro inhibitors. The toxic dose of compound 4 was more than 80 µM for 24, 48, and 10 72 h. Compound 4 showed dose-dependent inhibition against HCV replication with a 11 considerable reduction in Rluc activity at 40 µM. This finding was further investigated by 12 immunostaining that showed the inhibitory effect of compound 4 against HCV NS3-4Apro 13 was dose-dependent. This study identified a unique small molecule compound that could 14 lead to the development of HCV NS3-A4pro inhibitors and would be useful to develop highly 15 effective drugs for HCV infection based on HCV NS3-4A protease inhibition. 16 17 18 Keywords: Antiviral compounds, Quinazoline derivatives, Hepatitis C virus, NS3-4A serine 19 protease, HCV replicon assay. 20 21 22 23 24 25 26 the inhibitory potential of five unique quinazoline derivatives towards HCV NS3-4A serine 1 protease and their ability to inhibit HCV replication in a cell-based HCV replicon system. 2 Methods 3 Chemical synthesis 4 Compound synthesis and characterization were carried out as previously described (17]. In 5 brief, one equivalent of amino benzhydrazide (2.5 mmol) was dissolved in 50 ml of ethanol. 6 Two equivalent of substituted aromatic salicylaldehyde (5.0 mmol) was then added to this 7 solution in the presence of glacial acetic acid (added as a catalyst at 50-60℃). The reaction 8 mixture was refluxed for 2-3h. The yellow to brown precipitates of the compounds below 9 were formed during the reactions. 10 Expression and Purification of HCV Protease 11 Protein purification was conducted as previously described with modification[18,19]. The 12 long oligonucleotides spanning DNA sequences of HCV genotype 3 proteases (NS3-GSGS-4A 13 pro) were overlapped and extended using Klenow DNA polymerase (Invitrogen, USA). PCR 14 subsequently amplifies the designed full-sized DNA and cloned into E. coli expression vector, 15 PQE30. The recombinant plasmids were then isolated, and the purified plasmids were 16 sequenced to verify the sequence identity of the insert. E. coli harboring the recombinant 17 protease vector was cultured in 1 L of LB medium and expression was induced by addition of 18 1mM IPTG followed by an additional 4 hours incubation at 370C. The bacterial cells were 19 then harvested, lysed, and sonicated on ice. Recombinant protein in the supernatan...