A series of stilbenes has been prepared and tested for cytotoxicity in the five human cancer cell lines A-549 non-small cell lung, MCF-7 breast, HT-29 colon, SKMEL-5 melanoma, and MLM melanoma. The cis stilbenes 6a-f proved to be cytotoxic in all five cell lines, with potencies comparable to that of combretastatin A-4. These cytotoxic compounds were all potent inhibitors of tubulin polymerization. The corresponding trans stilbenes 7b-f were inactive as tubulin polymerization inhibitors and were significantly less cytotoxic in the five cancer cell lines. In the dihydro series, 8b, 8c, and 8f were inactive as tubulin polymerization inhibitors, while 8a, 8d, and 8e were less active than the corresponding cis compounds 6a, 6d, and 6e. The lack of tubulin polymerization inhibitory activity and cytotoxicity displayed by the phenanthrene 23b, which was synthesized as a conformationally rigid analogue of the lead compound 1, indicates that the activity of the stilbenes is not due to a totally planar conformation. Similarly, inactivity of the conformationally restricted analogue 26 suggests that the biologically active conformation of 1a resembles that of the cis alkene 1. Additional inactive compounds prepared include the benzylisoquinoline series 28-32 as well as the protoberberines 38 and 39. Shortening the two-carbon bridge of 1a to a one-carbon bridge in the diphenylmethane 20 resulted in a decrease in cytotoxicity and tubulin polymerization inhibitory activity. Although the corresponding benzophenone 18 was as active as 1a as a tubulin polymerization inhibitor, it was less cytotoxic than 1a, and the benzhydrol 19 was essentially inactive. With the exception of the amide 15c, which displayed low antitubulin activity, all of the phenylcinnamic acid derivatives 14a-c and 15a-f were inactive in the tubulin polymerization inhibition assay. The acid 14b and the ester 15a were cytotoxic in several of the cancer cell cultures in spite of their inactivity as tubulin polymerization inhibitors.

In order to define the structural parameters associated with the antitubulin activity and cytotoxicity of 2-methoxyestradiol, a mammalian metabolite of estradiol, an array of analogs was synthesized and evaluated. The potencies of the new congeners as inhibitors of tubulin polymerization and colchicine binding were determined using tubulin purified from bovine brain, and the cytotoxicities of the new compounds were studied in a variety of cancer cell cultures. Maximum antitubulin activity was observed in estradiols having unbranched chain substituents at the 2-position with three non-hydrogen atoms. 2-Ethoxyestradiol and 2-((E)-1-propenyl)-estradiol were substantially more potent than 2-methoxyestradiol itself. The tubulin polymerization inhibitors in this series displayed significantly higher cytotoxicities in the MDA-MB-435 breast cancer cell line than in the other cell lines studied. The potencies of the analogs as cytotoxic and antimitotic agents in cancer cell cultures correlated with their potencies as inhibitors of tubulin polymerization, supporting the hypothesis that inhibition of tubulin polymerization is the mechanism of the cytotoxic action of 2-methoxyestradiol and its congeners. Several of the more potent analogs were tested in an estrogen receptor binding assay, and their affinities relative to estradiol were found to be very low.

A new series of estradiol analogs was synthesized in an attempt to improve on the anticancer activity of 2-methoxyestradiol, a naturally occurring mammalian tubulin polymerization inhibitor. The compounds were evaluated as inhibitors of tubulin polymerization and the binding of [3H]colchicine to tubulin, as well as for in vitro cytotoxicity in human cancer cell cultures. Overall, the most potent of the new compounds were 2-(2',2',2'-trifluoroethoxy)-6-oximinoestradiol, 2-ethoxy-6-oximinoestradiol, and 2-ethoxy-6-methoximinoestradiol. These agents lacked significant affinity for the estrogen receptor. The cytotoxicities of the compounds correlated in general with their abilities to inhibit tubulin polymerization, thus supporting inhibition of tubulin polymerization as the primary mechanism causing inhibition of cell growth.

Although certain substituted cis-stilbenes have displayed potent tubulin polymerization inhibitory activity and significant cytotoxicities in cancer cell cultures, these compounds have limited aqueous solubility and are therefore difficult to formulate for in vivo evaluation. A series of water-soluble N-(3,4,5-trimethoxybenzyl)aniline salts has therefore been synthesized in which the olefinic bridge of the stilbenes is replaced by an aminomethylene hydrochloride moiety. A relationship was found between the size of the substituent in the 4-position of the aniline ring and both antitubulin activity and cytotoxicity, such that the smaller the substituent, the greater the potency. The most promising of the newly synthesized compounds was 4-methyl-N-(3,4,5-trimethoxybenzyl)aniline hydrochloride, with an IC50 value of 3.5 microM for inhibition of tubulin polymerization and cytotoxicity for a wide variety of cancer cell lines. The cytotoxicities of the benzylaniline hydrochlorides correlated remarkably well with their antitubulin activities.

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