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Abstract

The dengue, West Nile and other orthoflaviviruses are emerging pathogens of global significance. There is no antiviral treatment available to treat infections with these viruses. The flaviviral NS2B-NS3 protease is one of the most promising targets for anti-flaviviral drug discovery. Recently developed peptidomimetic compounds could overcome pharmacokinetic liabilities of previous inhibitors related to charge and polarity but, in turn, had become non-drug-like due to high lipophilicity and molecular weight, and their risk of racemisation. Another substantial impairing factor in the antiflaviviral drug development is the lacking predictive power for cellular activity of the commonly used biochemical assays on the isolated protease. This thesis presents the discovery, development and optimisation of novel heterocyclic classes of flaviviral protease inhibitors including their design, synthesis, analytical characterisation and biological evaluation in biochemical and cellular assays. Besides, novel conditions for the biochemical isolated dengue and West Nile virus protease assays are presented that are more similar to in vivo conditions and show substantially improved predictability of cellular activity for a broad set of compounds compared to previous widely used assay conditions. The inhibitors presented herein are proline and pyrazole-3-carboxylic acid derivatives – compound classes commonly known in various approved drugs. Inhibitors of both classes show low micromolar or even submicromolar activity in a cellular DENV-2 protease reporter gene assay as well as in an immunofluorescence-based DENV-2 antiviral assay. They also show promising activity in the revised biochemical assays down to single-digit micromolar IC50 values. Enzyme-kinetic studies indicate active site binding and mixed competitive inhibition of the dengue virus protease. The compounds have negligible off-target inhibition, no cytotoxicity in HeLa cells at relevant concentrations and are stable against rat liver microsomes. A robust synthetic route towards pyrazole-3-carboxylic acid derivatives based upon the traditional Knorr pyrazole synthesis was established to enable rapid synthesis of novel inhibitors with structurally diverse features. Introduction of a pyrrolidinylpyrimidine moiety led to the most active compounds presented in this thesis. Extensive structure-activity relationship studies reveal an encouraging perspective for the further development of drug-like small molecules to combat flaviviral infections.