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

Abstract

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(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 (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 (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 IC(50) 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.

Figure 1

First example of library development that employs the indole aryne methodology to construct 6,7-annulated-4-substituted indole compounds. Top: Bartoli route to prepare the N-methyl-4,6,7-tribromoindole scaffold. Bottom: Selective generation of 4-bromo-6,7-indole aryne, and strategic combination of 6,7-indolyne cycloaddition and cross-coupling reactions under both Suzuki-Miyaura and Buchwald-Hartwig conditions to construct polycyclic indole libraries.

Figure 2

Chemical structures and identification numbers of the most effective antiproliferative 6,7-annulated-4-substituted indole compounds tested for their antitumor effects in L1210 cells in vitro.

Figure 3

Comparison of the ability of serial concentrations (plotted on a logarithmic scale) of KU-69 (○, ●), KU-87 (□, ■) and KU-95 (△, ▲) to inhibit the metabolic activity of L1210 tumor cells at days 2 (open symbols) and 4 (solid symbols) in vitro. L1210 cell proliferation results were expressed as % of the net absorbance of MTS/formazan after bioreduction by vehicle-treated control cells after 2 (A_{490 nm}=1.112±0.048) and 4 (A_{490 nm}=1.177±0.051) days in culture (100±4.3%, striped area). The blank values (A₄₉₀ nm=0.432 at day 2 and 0.434 at day 4) for cell-free culture medium supplemented with MTS:PMS reagent were subtracted from the results. Bars: means±SD (n=3). ^aNot different from respective controls; ^bp<0.05, ^cp<0.01 and ^dp<0.005, lower than respective controls.

Figure 4

Comparison of the ability of serial concentrations (plotted on a logarithmic scale) of KU-70 (○, ●), KU-80 (□, ■) and KU-96 (△, ▲) to inhibit the metabolic activity of L1210 tumor cells at days 2 (open symbols) and 4 (solid symbols) in vitro. The conditions of the experiment and the determination of the results were identical to those of Figure 3. Vehicle-treated control cells after 2 and 4 days: 100±4.3%, striped area. Bars: means±SD (n=3). ^aNot different from respective controls; ^bp<0.05, ^cp<0.025, ^dp<0.01 and ^ep<0.005, lower than respective controls.

Figure 5

Comparison of the ability of 25 μM concentrations of the antiproliferative KU-69, KU-70, KU-72, KU-80, KU-87, KU-95, KU-96, KU-113 and KU-191 compounds to inhibit the rate of incorporation of [³H]thymidine into DNA measured in L1210 cells over 30 min following a 3-h period of incubation at 37°C in vitro. The magnitude of DNA synthesis inhibition caused by 0.256 μM mitoxantrone (Mitox) was used as a positive control. DNA synthesis in vehicle-treated control cells (C) at 37°C was 20,387±1,325 cpm (100±6.5%). The blank value (766±43 cpm) for control cells incubated and pulse-labeled at 2°C with 1.5 μCi of [³H]thymidine has been subtracted from the results. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.025, less than control.

Figure 6

Comparison of the ability of serial concentrations (logarithmic scale) of the antiproliferative KU-70 (●) and KU-80 (○) compounds to inhibit the rate of incorporation of [³H]thymidine into DNA measured in L1210 cells over 30 min following a 3-h period of incubation at 37°C in vitro. The conditions of the experiment and the determination of the results were identical to those of Figure 5. DNA synthesis in vehicle-treated control cells at 37°C: 100±6.5%, striped area. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.05 and ^cp<0.025, less than control.

Figure 7

Comparison of the ability of 8 μM mitoxantrone (Mitox), 50 μM actinomycin D (Act-D), 125 μM m-amsacrine (Amsa) and serial concentrations of antiproliferative KU-80 compound to inhibit the binding of EB to double-stranded ctDNA. Results were expressed as % of the control (C) fluorescence of the EB-DNA complex in the absence of drug at 525 nm excitation and 600 nm emission (162.1±5.3 arbitrary units; 100±3.3 %). The background of EB fluorescence in the absence of DNA (26.3±1.0 arbitrary units) was subtracted from the results. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.05, ^cp<0.025 and ^dp<0.005, smaller than control.

Figure 8

Comparison of the ability of 25 μM concentrations of the antiproliferative KU-69, KU-70, KU-72, KU-80, KU-87, KU-95, KU-96, KU-113 and KU-191 compounds to induce DNA fragmentation at 24 h in L1210 cells containing 3H-prelabeled DNA in vitro. The levels of DNA fragmentation caused by 1.6 μM daunorubicin (DAU) and 0.256 μM mitoxantrone (Mitox) were used as positive controls. The results were expressed as [cpm in supernatant/(cpm in supernatant + pellet)] × 100 at 24 h. For vehicle-treated control (C) tumor cells (3.83±0.84% DNA fragmentation), the supernatant (DNA fragments) was 2,096±126 cpm and the pellet (intact DNA) was 52,628±3,368 cpm. Bars: means±SD (n=3). ^ap<0.05 and ^bp<0.025, greater than control.

Figure 9

Comparison of the ability of serial concentrations (logarithmic scale) of the antiproliferative KU-80 (○), KU-96 (□) and KU-191 (△) compounds to induce DNA fragmentation at 24 h in L1210 cells, containing ³H-prelabeled DNA in vitro. The conditions of the experiment and the determination of the results were identical to those in Figure 8. DNA fragmentation in vehicle-treated control tumor cells: 3.83±0.84%, striped area. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.05 and ^cp<0.025, greater than control.

Figure 10

Fluorogenic assay of effector caspase activation in drug-treated L1210 cells. Comparison of the time-dependent inductions of caspase-3-like protease activity by 0.64 μM mitoxantrone (▲, Mitox), 1.6 μM daunorubicin (□, DAU), 62.5 μM KU-80 (○) and 62.5 μM KU-96 (●) in L1210 cells, incubated for 3–24 h in vitro. Results are expressed as percentage of DEVD cleavage activity in vehicle-treated control tumor cells (4.06±0.36 nmol AFC released/mg protein, 100±8.9%, striped area) at each time point tested. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.025 and ^cp<0.005, greater than control.

Figure 11

Comparison of the effects of the antiproliferative KU-69 compound, vincristine (VCR) and taxol on the frequency of mitotic figures (A) and binucleated cells (BNCs) (B) in L1210 tumor cells in vitro. L1210 cells were incubated in triplicate for 24 (open columns) and 48 h (closed columns) at 37°C in the presence, or absence (C: control) of the indicated concentrations of drugs. The percentage of cells in each category was determined by morphologic analysis, scoring at least 2,000 cells/slide to identify those containing mitotic figures or 2 nuclei. Results were expressed as % of mitotic (A) or BNCs (B) in drug-treated cultures divided by the % of mitotic (C: 0.42±0.08%) or BNCs (C: 2.73±0.57%) in vehicle-treated controls. Bars: means±SD (n=3). ^ap<0.05, smaller than control; ^bp<0.01 and ^cp<0.025, greater than respective controls.

Figure 12

Comparison of the effects of the antiproliferative KU-69 compound, vincristine (VCR) and taxol on the frequency of multinucleated (MULTI) cells (A) and micronuclei (MNi) (B) in L1210 tumor cells in vitro. L1210 cells were incubated in triplicate for 24 (open columns) and 48 h (closed columns) at 37°C in the presence or absence (C: control) of the indicated concentrations of drugs. The percentage of cells in each category was determined by morphological analysis, scoring at least 2,000 cells/slide to identify those containing 3–4 nuclei or MNi. A: Results were expressed as % of MULTI cells in drug-treated cultures divided by the % of MULTI cells in vehicle-treated controls (C: 0.34±0.07%). B: Results were expressed as % of vehicle- (C: 0.06±0.01%) or drug-treated cells with MNi. Bars: means±SD (n=3). ^aNot different from control; ^bp<0.005, greater than respective controls.