Four new 5-substituted camptothecins (4-7) have been synthesized and evaluated for DNA topoisomerase I inhibition. The results suggested that the pyridone moiety in the D ring of camptothecin plays a crucial role in determining its activity and that the 5 position of the C ring should be unsubstituted for retention of activity. The early structure-activity relationship studies 2, 3 of camptothecin (1) showed that the structural requirements for antitumor activity include the presence of the planar ABCD ring system and the β-hydroxy lactone moiety of ring E. Substitution of ring A with small groups, such as 10-hydroxy, is also permissible, with many of these analogs being considerably more active than 1. In the search for clinically useful camptothecin analogs, a number of derivatives have been synthesized based on the above knowledge. In the design of these analogs, much effort has been directed towards introducing a water-soluble moiety into the 9 or 10 position of the A ring of the camptothecin. The most successful examples are the 7,10-substituted analog CPT-11 and the 9-substituted analogs Topotecan and 9-amino camptothecin, which are being tested clinically as anticancer drugs against colon and other cancers in Japan, 4 Europe, 5 and the U.S.A., 6, 7 respectively. In our previous report, a series of novel water-soluble 7-(aminoacylhydrazono)-formyl camptothecins with increased topoisomerase I inhibitory activities were synthesized. Our results suggested that the 7 position of the B ring is a suitable location for introducing a polar moiety into camptothecin to produce analogs with enhanced topoisomerase I inhibitory activity. 8, 9 As an extension to this study, an investigation of the 5-substituted camptothecins was undertaken. A few 5-substituted camptothecins have been reported; however, no biological data are available.10 The generation of α,β-epimers has also made this position more difficult to manipulate. On the other hand, the significance of the pyridone moiety of the D ring on camptothecin's activity is still unclear. To explore the practicality of active 5-substituted camptothecins and to clarify the significance of the pyridone moiety, we have synthesized four new 5-substituted camptothecins and have evaluated their inhibitory effects on DNA topoisomerase I.