Quinoxalinehydrazines represent a novel class of compounds with excellent potency in a panel of cancer cell lines. A prototype compound, SC144, showed significant in vivo efficacy in mice xenograft models of human breast cancer cells. The subsequent structure-activity relationship study resulted in the discovery of SC161 with better potency in cancer cell lines. Further exploring the possible conformational space by a 10 ns molecular dynamics simulation as presented herein, resulted in various pharmacophore orientations. The trajectory analysis indicated that in most of the simulation time, the molecule stays favorably in a compact planarlike orientation. We therefore built a pharmacophore model based on the cluster containing the highest number of frames to represent the most probable orientation. The model was used to screen a subset of our small molecule database containing 350,000 compounds. We selected 35 compounds for the initial cytotoxicity screen. Seventeen compounds belonging to oxadiazolopyrazine and quinoline class displayed cytotoxicity in various cancer cell lines. Five of them, compounds 2, 6, 15, 16, and 19, all bearing an oxadiazolopyrazine scaffold, showed IC(50) values <3 muM in certain tumor cell lines. The most potent compound, 2, showed IC(50) values <2 muM in HCT116 p53(+/+), HCT116 p53(-/-), and HEY cells, and 8 muM in NIH3T3 cells. This study shows that conformational sampling of a lead small molecule followed by representative pharmacophore model development is an efficient approach for the rational design of novel anticancer agents with similar or better potency than the original lead but with different physicochemical properties.