are several anti-hepatitis C virus agents in clinical trials. Structure–activity relationship Nucleoside Inhibitors Nucleoside inhibitors of hepatitis C virus NS5B are classified into three major classes: purine nucleoside, inhibitors, pyrimidine nucleoside inhibitors, and miscellaneous nucleoside inhibitors. Sofosbuvir is a pyrimidine nucleoside analog and a phosphoramidite prodrug. Pyrimidine nucleoside inhibitors The isomeric form of the amino acid matters since the D-alanine derivative is inactive which means that the natural L-amino acid is required for activity. When observing the amino acid side chain (R1) a small alkyl group is a viable substitution, but the potency reduces significantly if the substitution is larger than ethyl. Methyl has the greatest potency and is, therefore, a viable substitution. When the amino acid is alanine and the phosphate ester is phenyl substituent the groups at the carboxylic acid ester (R2) that provide the desired submicromolar activity are small alkyl groups and branched alkyl groups, for example, methyl, ethyl, isopropyl, n-butyl and 2-butyl. But cytotoxicity is observed with n-butyl, 2-butyl and n-pentyl esters. Phenyl and halogenated alkyl groups do not provide enough potency enhancement. When evaluating the phosphoramidate ester substituent (R3) the 1-naphthyl ester has the greatest potency but it is also cytotoxic so it is not a viable substituent. Mono- and dihalogenerated phenolic esters also have good potency but they are also cytotoxic. A derivative with phenol as a substituent has good potency and is not cytotoxic. The cyclohexyl ester derivatives make the most difference in the structure activity relationship as they show as much as 10-fold improvement in potency. Non-nucleoside Inhibitors Based on different binding site characteristics, non-nucleoside inhibitors of hepatitis C virus NS5B are classified into three distinct categories: active site inhibitors, allosteric site inhibitors, and miscellaneous non-nucleoside inhibitors. For miscellaneous non-nucleoside analogue inhibitors, the binding site is either not defined or under further investigation. Allosteric site inhibitors Allosteric site inhibitors are a large group within the non-nucleoside category with at least 10 different analogs that are known. One of these allosteric inhibitors is N-benzoyl pyrrolidine. In 2005 GlaxoSmithKline identified N-benzoyl pyrrolidine as a NS5B inhibitor through high-throughput screening. An isobutyl analog (table 2) showed IC50=0.7 μM submicromolar activity against NS5B. In the development, 5-phenyl was replaced with different heterocycles to try to optimize the inhibition, compounds (2-4). The 2-pyridyl (4) group was well tolerated on the pyrrolidine scaffold, whereas replacement with 2-thienyl led to compound (5) with IC50= 0,3 μM. To get a