In order to develop potential backup compounds for our previous identified new anticancer drug candidate,CHM-l-P-Na, a series of 6-alkylamino-2-(3-hydroxyphenyl)quinolin-4-one derivatives were designed and synthesized. The newly synthesized derivatives were evaluated for cytotoxicity and structure-activity relationships were established. Among those tested compounds, 6-pyrrolidino-2-(3-hydroxyphenyl)quinolin-4-one (A) was the most promising agent which was submitted to NCI for evaluation against 60 human cancer cell lines. Furthermore, the monophosphate of compound A (A-P) was synthesized and evaluated for antitumor activity in Hep3B Xenograft nude mice model. The results indicated that compound A-P has excellent antitumor activity when administrated either iv or po. With excellent antitumor activity profiles, compound A-P is considered potential backup compound for CHM-l-P-Na, which is currently under preclinical study.