J. Med. Chem. dimethylformamide (1 mL) was treated with 60% sodium hydride in oil (20 mg) for 20 min, and then with ethyl iodide (0.02 mL) for 20 min longer. The mixture was concentrated to dryness at 0.1 mm, taken up in ethyl acetate, washed with sodium bicarbonate solution, dried (Mg#O$, concentrated to dryness, and triturated with ether to afford the ester as an off-white solid, mp 110-118 OC (30 mg, 31%). Similarly, 3s was prepared from 3a i991,34, m 9 -2 5~ 2579 through the use of tert-butyl 4-(2-aminoethyl)phenylacetate.% An array of cis-, trans-, and dihydrostilbenes and some N-arylbenzylamines were synthesized and evaluated for their cytotoxicity in t.he five cancer cell cultures A-549 lung carcinoma, MCF-7 breast carcinoma, HT-29 colon adenocarcinoma, SKMEL-5 melanoma, and MLM melanoma. Several cis-stilbenes, structurally similar to combretastatins, were highly cytotoxic in all five cell lines and these were also found to be active as inhibitora of tubulin polymerization. The most active compounds also inhibited the binding of colchicine to tubulin. The most potent of the new compounds, both as a tubulin polymerization inhibitor and as a cytotoxic agent, was (Z)-l-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene (Sa). This substance was almost as potent as combretastatin A-4 (la), the most active of the combretastatins, as a tubulin polymerization inhibitor. Compound 5a was found to be approximately 140 times more cytotoxic against HT-29 colon adenocarcinoma cells and about 10 times more cytotoxic against MCF-7 breast carcinoma cells than combretastatin A-4. However, 5a was found to be about 20 times less cytotoxic against A-549 lung carcinoma cells, 30 times less cytotoxic against SKMEL-5 melanoma cells, and 7 times less cytotoxic against MLM melanoma cells than combretastatin A-4. The relative potencies 5a > 8a > 6a for the cis, dihydro, and trans compounds, respectively, as inhibitors of tubulin polymerization are in agreement with the relative potencies previously observed for combretastatin A-4 (la), dihydrocombretastatin A-4 (IC), and transcombretastatin A-4 (lb). The relative potencies 5a > 8a > 6a were also reflected in the results of the cytotoxicity assays. Structure-activity relationships of this group of compounds are also discussed.

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.

Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those druginduced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show crossresistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G 2 -M and a dependence on functional p53 for their cytotoxicity. Dose-dependent ;-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These ;-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0). [Cancer Res 2007; 67(21):10397-405]

A number of indenoisoquinolines were prepared and evaluated for cytotoxicity in human cancer cell cultures and for activity vs topoisomerase 1 (top1). The two most cytotoxic indenoisoquinolines proved to be cis-6-ethyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (21) and cis-6-allyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22), both of which displayed submicromolar mean graph midpoints when tested in 55 human cancer cell cultures. Two of the most potent top1 inhibitors were 6-(3-carboxy-1-propyl)-5,6-dihydro-5, 11-dioxo-11H-indeno[1,2-c]isoquinoline (26) and 6-ethyl-2, 3-dimethoxy-8,9-(methylenedioxy)-11H-indeno[1,2-c]isoquinolinium chloride (27), both of which also inhibited top2, unwound DNA, and are assumed to be DNA intercalators. However, two additional potent top1 inhibitors, 6-allyl-5,6-dihydro-2,3-dimethoxy-8, 9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13c) and 5,6-dihydro-6-(4-hydroxybut-1-yl)-2,3-dimethoxy-8, 9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (19a), did not unwind DNA and did not affect top2. Some of the DNA cleavage sites detected in the presence of the indenoisoquinolines were different from those seen with the camptothecins. The cleavage sites induced by the indenoisoquinolines were reversed by salt treatment, which is consistent with the reversible trapping of top1 cleavable complexes by the indenoisoquinolines. In general, the potencies of the indenoisoquinolines as top1 inhibitors did not correlate with their potencies as cytotoxic agents, as some of the most cytotoxic agents had little if any effect on top1. On the other hand, the most potent of the indenoisoquinolines vs top1 were not the most cytotoxic. In several cases, moderate activity was observed for both cytotoxicity and activity vs top1.