Discovery of Pyrazole-3-Carboxylic Acid Derivatives as Dengue Virus Protease Inhibitors with Antiviral Activity

We present the discovery of pyrazole-3-carboxylic acid derivatives as novel dengue virus (DENV) NS2B-NS3 protease inhibitors. The discovery was triggered by omission of the phenylglycine scaffold of previous lead structures. We established a robust, regioselective synthetic route toward pyrazole-3-carboxylic acid derivatives. Subsequent SAR studies delivered inhibitors with promising activity against the DENV protease in biochemical and reporter gene assays with EC₅₀ values down to 2.2 μM and antiviral activity against DENV-2 with EC₅₀ values down to 4.1 μM. Active site binding and target specificity were evaluated by a tryptophan fluorescence quenching assay. We further observed negligible cytotoxicity, no inhibition of the off-targets thrombin and trypsin, and promising early stage pharmacokinetic properties. The 2-aminopyrimidine scaffold was identified as a promising nonbasic replacement of the guanidine moiety. In addition, eliminating the highly hydrophobic phenylglycine moiety of previous compound series provides a crucial increase in drug likeness of this novel flaviviral protease inhibitor class.