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

Indole arynes, or indolynes, represent a new class of reactive aryne intermediate (1–7) but the biological properties of annulated indoles have not been fully-evaluated and exploited. However, a few biologically-active natural products such as trikentrins, herbindoles, teleocidins and nodulisporic acids have been reported (8–11), suggesting that annulated indoles may have unique structural features and a good probability of exhibiting interesting biological activity (12). Because annulated indoles have almost no representation in the NIH PubChem and Molecular Library Small Molecule Repository (MLSMR) databases, an unprecedented class of an indole-based library was constructed, using the indole aryne methodology (12), and screened for antitumor activity (13). Hence, 66 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 (12), and tested for their effectiveness against murine L1210 tumor cell proliferation in vitro (13). Most compounds inhibited the metabolic activity of L1210 lymphocytic leukemia cells in a time- and concentration-dependent manner but only nine of them were sufficiently potent to inhibit L1210 tumor cell proliferation by 50% in the low micromolar range after two [concentration inhibiting by 50% (IC50)=4.5–20.4 μM] and four days [IC50=0.5–4.0 μM] in culture (13). 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 3H-thymidine (13). 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 (13). However, all nine antiproliferative compounds induced DNA cleavage at 24 h in L1210 cells that contained 3H-thymidine-prelabeled DNA, suggesting that these antitumor-annulated indoles might trigger an apoptotic pathway of DNA fragmentation (13). Indeed, these annulated indoles caused a time-dependent increase of caspase-3 activity with a peak at 6 h (13). Interestingly, antiproliferative concentrations of annulated indoles increased the mitotic index of L1210 cells and stimulated the formation of many bi-nucleated cells (BNCs), multi-nucleated cells (MNCs), apoptotic cells and micronuclei (MNi) after 24-48 h, suggesting that these antitumor compounds might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis to induce apoptosis (13). Therefore, the present study was undertaken to determine the effectiveness of the six most potent annulated indoles against human HL-60 tumor cell proliferation, assess their ability to alter the kinetics of tubulin and actin polymerization, and compare their effects to those of drugs known to interact with microtubules (MTs) and actin filaments in order to disrupt the functions of the mitotic spindle and cleavage furrow.