Abstract Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5–6 steps in 25–45% overall yields. Their structures were confirmed by 1H NMR and 13C NMR as well as LC–MS. The anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2 and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency. This study leads to the identification of two highly active molecules, compounds 2h (IC50=3nM) and 2k (IC50=6nM), against human leukemia K562 cells.