Design, Synthesis, and Biological Evaluation of Substituted 4,6-Dihydrospiro[[1,2,3]triazolo[4,5‑<i>b</i>]pyridine-7,3′-indoline]-2′,5(3<i>H</i>)‑dione Analogues as Potent NS4B Inhibitors for the Treatment of Dengue Virus Infection

A series of substituted 4,6-dihydrospiro­[[1,2,3]­triazolo­[4,5-<i>b</i>]­pyridine-7,3′-indoline]-2′,5­(3<i>H</i>)-dione analogues were synthesized and evaluated as potent dengue virus inhibitors. Throughout a structure–activity relationship exploration on the amide of the indolone moiety, a wide range of substitutions were found to be well tolerated for chemical optimization at this position. Among these compounds, <b>15</b> (<b>JMX0254</b>) displayed the most potent and broad inhibitory activities, effective against DENV-1 to -3 with EC₅₀ values of 0.78, 0.16, and 0.035 μM, respectively, while compounds <b>16</b>, <b>21</b>, <b>27</b>–<b>29</b>, <b>47</b>, and <b>70</b> exhibited relatively moderate to high activities with low micromolar to nanomolar potency against all four serotypes. The biotinylated compound <b>73</b> enriched NS4B protein from cell lysates in pull-down studies, and the findings together with the mutation investigations further validated dengue NS4B protein as the target of this class of compounds. More importantly, compound <b>15</b> exhibited good <i>in vivo</i> pharmacokinetic properties and efficacy in the A129 mouse model, indicating its therapeutic potential against the dengue virus infection as a drug candidate for further preclinical development.