Thymidine phosphorylase (TPase) is one of the key enzymes involved in the pyrimidine nucleoside salvage pathway. However, TPase also stimulates angiogenesis, and its expression correlates well with microvessel density and metastasis in a variety of human tumors. We have shown recently that 5'-O-trityl-inosine (KIN59) allosterically inhibits TPase enzymatic activity. KIN59 also inhibits TPase-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay. The trityl group was found to be instrumental to preserve both the anti-TPase and antiangiogenic effect. We have now synthesized a variety of novel 5'-O-trityl nucleoside derivatives. Enzyme activity studies showed that the anti-TPase activity is significantly improved by replacement of the hypoxanthine base by thymine [3.5-fold; i.e., 5'-O-tritylthymidine (KIN6)] and the introduction of chloride on the trityl group [7-fold; i.e., 5'-O-(4-chlorotrityl)-inosine (TP136)], whereas removal of 2'-hydroxyl in the ribose did not significantly alter the anti-TPase activity. Enzyme kinetic studies also demonstrated that 1-(5'-O-trityl-beta-d-ribofuranosyl)-thymine (TP124), like KIN59, inhibits TPase in a noncompetitive fashion both with respect to phosphate and thymidine. Most KIN59 analogs markedly inhibited TPase-induced angiogenesis in the CAM assay. In vitro studies showed that the antiangiogenic effect of these compounds is not attributed to endothelial cell toxicity. For several compounds, there was no stringent correlation between their anti-TPase and antiangiogenic activity, indicating that these compounds may also act on other angiogenesis mediators. The antiangiogenic 5'-O-trityl nucleoside analogs also caused degradation of pre-existing, immature vessels at the site of drug exposure. Thus, 5'-O-trityl nucleoside derivatives combine antiangiogenic and vascular-targeting activities, which opens perspectives for their potential use as anticancer agents.