DNA Topoisomerase I (TopI) is over-expressed in tumor cells and is an clinical important target for a variety of caner chemotherapies. Herein, we have identified the antiproliferative activity of some sulphonic acid esters derived from rutaecarpine as well as 5-methylene rutaecarpine, for the purpose of improving therapeutic benefits of camptothecin and evodiamine. The synthesized N13-substituted rutaecarpine compounds were evaluated for their in vitro cytotoxicity against A549, HepG-2, U251, HeLa and MCF-7 human carcinoma cell lines by MTT assay, of which the hit 3d exhibited potent anti-tumor activities on all cell lines. Additionally, 3d was found to inhibit substantially the tumor growth on the HepS-bearing mice at a dose of 80 mg/kg. Subsequently, preliminary structure-activity relationship was explored based on the combination of biological data and ligand-based molecular modeling methods, which could provide guidance for designing new analogues of rutaecarpine. Finally, in silico screening studies of sulfonic rutaecarpine esters revealed that they could form hydrogen-bonding and hydrophobic interactions with several amino acid residues of topoisomerase I at the cleavage site, resembling the binding format between camptothecin and topoisomerase I-DNA complex.