Dengue virus (DENV) causes about 50-100 million cases per year worldwide. However, there is still a big challenge in developing antiviral drugs against DENV infection. Some derivatives of alkaloid (-)-cytisine, like other alkaloid analogs, have been proposed for their antiviral potential. This study investigated antiviral activity and mechanisms of the cytisine derivatives, and discovered the structure-activity relationship against DENV. The antiviral assays were performed using one strain of DENV1 and DENV2, and two cell lines Vero E6 and A549. The structure-activity relationship of the effective compounds was also evaluated using combination of time-of-addition/removal assay and molecular docking. Compounds 3, 4, 12 (N-allylcytisine-3-thiocarbamide), 16, and 20 exhibited the high antiviral activity with IC50 values of lower than 3 μM against DENV1 and DENV2. Of them, the derivative 12 showed the highest antiviral activities against DENV1 (IC50 = 0.14 μM) and DENV-2 (IC50 =
