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Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. Here, we describe new 1-(ω-phenoxyalkyl)uracils bearing acetanilide fragment in 3 position of pyrimidine ring as potential antiviral drugs against HCV. Using a combination of various biochemical assays and in vitro virus infection and replication models, we show that our compounds are able to significantly reduce viral genomic replication, independently of virus genotype, with their IC₅₀ values in the nanomolar range. We also demonstrate that our compounds can block de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds. A detailed structure-activity relationship revealed that the most active compounds were the N³-substituted uracil derivatives …