(+)-Rutamarin inhibits EBV lytic DNA replication with an IC50 of 7.0 μM. (-)-Chalepin, a (-)-rutamarin derivative, was isolated from the whole plant of Ruta graveolens and used as a precursor of (-)-rutamarin. Altogether, 28 (-)-rutamarin derivatives were synthesized starting from (-)-chalepin. Of these, 16 compounds (2a-e, 3b-e, 3g, 4f, 4k, 4m-p) were found to be more potent against EBV lytic DNA replication than (-)-chalepin. Compounds 4m, 4n, and 4p exhibited IC50 values of 1.5, 0.32, and 0.83 μM and showed selectivity index values (SI) of 801, 211, and >120, respectively. Thus, compounds 4m, 4n, and 4p are considered promising leads for further laboratory investigation.