Abstract: Starting with natural anticancer product podophyllotoxin, its C-ring was modified in five steps. Podophyllotoxin was converted into important intermediate product 4β-isothiocyanate-4-deoxypodophyllotoxin through azide, reduction, addition, and elimination, respectively. Then it reacted with a series of hydrazide compounds to obtain various thiosemicarbazide derivatives, which were finally cyclized to afford a series of 4β-(1, 3, 4-oxadiazole-2-amino)-podophyllotoxin derivatives. Previous studies had shown that when R group of 4β-(1, 3, 4-oxadiazole-2-amino)-podophyllotoxin derivatives was methyl, the biological activity has been more potent than podophyllotoxin against HepG2 and HeLa cell lines. Furthermore, it had also exhibited much lower cytotoxicity toward the normal cell lines L929 and Vero than etoposide, podophyllotoxin and fluorouracil. On this basis, a series of novel compounds with R group of linear chain alkyl or cycloalkanes were designed and synthesized to explore compounds with better antitumor activity, higher selectivity, and lower cytotoxicity. Their cytotoxicity in vitro against six tumor cell lines Du-145, HeLa, A549, K562, K562/adr and HepG2 was evaluated by standard MTT assay. Experimental results showed that compounds 6a, 6b and 6d had significant antitumor activity.