A series of 9-(alkylthiomethyl)-10-hydroxycamptothecins and pyrano-fused camptothecin derivatives were synthesized via the reaction of topotecan hydrochloride with various thiols and alkyl vinyl ethers respectively. In the reactions, topotecan hydrochloride was utilized as ortho-quinonemethide (o-QM) precursor. The configuration of 19 was determined by (1)H NMR and NOESY spectra as syn-isomers, suggesting that the cycloaddition of topotecan with alkyl vinyl ethers could undergo a hetero Diels-Alder reaction. All the synthesized compounds were screened on cancer cell lines HepG2, KB, HCT-8 and SGC7901. Some compounds were selected to assess their inhibitory activity against Topo I via Topo I mediated DNA cleavage assays. The results showed that among those tested 9-(alkylthiomethyl)-10-hydroxycamptothecins, the compounds with bulkier hydrophobic side chains at 9-position have better bioactivities. As well as all pyrano-fused camptothecins possess antiproliferative activity against the tested cancer cell lines. Docking studies suggested that there are more interactions between the novel analogues and the binding site of Topo I.