A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the relationships between structure and activity. The compounds were designed to diminish the potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of tubulin polymerization and for cytotoxicity. 17alpha-methyl-beta-estradiol (30), 2-propynyl-17alpha-methylestradiol (39), 2-ethoxy-17-(1'-methylene)estra-1,3,5(10)-triene-3-ol (50) and 2-ethoxy-17alpha-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than 2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating metabolic processes. All of the compounds tested were cytotoxic in the panel of 55 human cancer cell cultures, and generally, the derivatives that displayed the most activity against tubulin were also the most cytotoxic.

A prodrug strategy was investigated to address the problem of limited aqueous solubility and the resulting limited bioavailability of the antitumor agent 2-methoxyestradiol. The 3-phosphate, 17-phosphate, and 3,17-diphosphate of 2-methoxyestradiol were synthesized. 2-methoxyestradiol 3-phosphate was metabolized more efficiently to the parent compound in vivo than 2-methoxyestradiol 17-phosphate, and it was also more cytotoxic in cancer cell cultures than either the 17-phosphate or the 3,17-diphosphate. These results agree with the in vivo anticancer activity of 2-methoxyestradiol 3-phosphate in a mouse Lewis lung carcinoma experimental metastasis model as opposed to the 17-phosphate and 3,17-diphosphate, both of which were inactive. The in vivo antitumor activity of 2-methoxyestradiol 3-phosphate at a dose of 200 mg/kg per day was comparable to that of a maximally tolerated dose of cyclophosphamide.