Antitumor effects of synthetic 6,7-annulated-4-substituted indole compounds in L1210 leukemic cells in vitro

Background: Because annulated indoles have almost no representation in the PubChem or MLSMR databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology, and screened for antitumor activity. Sixty-six novel 6,7-annulated-4-substituted indole compounds were synthesized, using a strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions, and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro. Materials and Methods: Various markers of tumor cell metabolism, DNA degradation, mitotic disruption, cytokinesis and apoptosis were assayed in vitro to evaluate drug cytotoxicity. Results: Most compounds inhibited the metabolic activity of leukemic cells in a time- and concentration-dependent manner but only 9 of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low-μM range after 2 (IC[subscript 50]: 4.5-20.4 μM) and 4 days (0.5-4.0 μM) in culture. However, the antiproliferative compounds that were the most effective at day 4 were not necessarily the most potent at day 2, suggesting different speeds of action. A 3-h treatment with antiproliferative annulated indole was sufficient to inhibit, in a concentration-dependent manner, the rate of DNA synthesis measured in L1210 cells over a 0.5-h period of pulse-labeling with [superscript 3]H-thymidine. Four of the antiproliferative compounds had weak DNA-binding activities but one compound reduced the fluorescence of the ethidium bromide-DNA complex by up to 53%, suggesting that some annulated indoles might directly interact with double-stranded DNA to disrupt its integrity and prevent the dye from intercalating into DNA base pairs. However, all 9 antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells, containing [superscript 3]H-thymidine-prelabeled DNA, suggesting that these antitumor annulated indoles might trigger an apoptotic pathway of DNA fragmentation. Indeed the antiproliferative annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h. Interestingly, the compounds with the most potent antiproliferative IC50 values at day 2 were consistently the most effective at inhibiting DNA synthesis at 3 h and inducing DNA fragmentation at 24 h. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells, multi-nucleated cells, apoptotic cells and micronuclei, suggesting that these antitumor compounds might enhance mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis. Conclusion: Although annulated indoles may have interesting bioactivity, novel derivatives with different substitutions must be synthesized to elucidate structure-activity relationships, identify more potent antitumor lead compounds, and investigate their molecular targets and mechanisms of action

Mechanisms by which synthetic 6,7-annulated-4-substituted indole compounds with anti-proliferative activity disrupt mitosis and block cytokinesis in human HL-60 tumor cells in vitro

Background: Synthetic 6,7-annulated-4-substituted indole compounds, which elicit interesting antitumor effects in murine L1210 leukemia cells, were tested for their ability to inhibit human HL-60 tumor cell proliferation, disrupt mitosis and cytokinesis, and interfere with tubulin and actin polymerization in vitro. Materials and Methods: Various markers of metabolic activity, mitotic disruption and cytokinesis were used to assess the effectiveness of the drugs in the HL-60 tumor cell system. The ability of annulated indoles to alter the polymerizations of purified tubulin and actin were monitored in cell-free assays and were compared to the effects of drugs known to disrupt the dynamic structures of the mitotic spindle and cleavage furrow. Results: With one exception, annulated indoles inhibited the metabolic activity of HL-60 tumor cells in the low-micromolar range after two and four days in culture but these anti-proliferative effects were weaker than those of jasplakinolide, a known actin binder that blocks cytokinesis. After 24-48 h, antiproliferative concentrations of annulated indoles increased the mitotic index of HL-60 cells similarly to vincristine and stimulated the formation of many bi-nucleated cells, multi-nucleated cells and micronuclei, similarly to taxol and jasplakinolide, suggesting that these antitumor compounds might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. Since annulated indoles mimicked the effect of vincristine on tubulin polymerization, but not that of taxol, these compounds might represent a new class of microtubule de-stabilizing agents that inhibit tubulin polymerization. Moreover, annulated indoles remarkably increased the rate and level of actin polymerization similarly to jasplakinolide, suggesting that they might also stabilize the cleavage furrow to block cytokinesis. Conclusion: Although novel derivatives with different substitutions must be synthesized to elucidate structure–activity relationships, identify more potent antitumor compounds and investigate different molecular targets, annulated indoles appear to interact with both tubulin to reduce microtubule assembly and actin to block cytokinesis, thereby inducing bi- and multinucleation, resulting in genomic instability and apoptosis

Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the Yellow Dyes Thioflavine S and Primuline

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (<b>P4</b>), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because <b>P4</b> also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound <b>17</b>) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM