3-[4-(1H-Indol-3-yl)-1,3-thiazol-2-yl]-1H-pyrrolo[2,3-b]pyridines, Nortopsentin Analogues with Antiproliferative Activity

A new series of nortopsentin analogues, in which the imidazole ring of the natural product was replaced by thiazole and the indole unit bound to position 2 of the thiazole ring was substituted by a 7-azaindole moiety, was efficiently synthesized. Two of the new nortopsentin analogues showed good antiproliferative effect against the totality of the NCI full panel of human tumor cell lines (~60) having GI50 values ranging from low micromolar to nanomolar level. The mechanism of the antiproliferative effect of these derivatives, investigated on human hepatoma HepG2 cells, was pro-apoptotic, being associated with externalization of plasma membrane phosphatidylserine and mitochondrial dysfunction. Moreover, the compounds induced a concentration-dependent accumulation of cells in the subG0/G1phase, while confined viable cells in G2/M phase.

More recently, hyrtinadine A, isolated from the marine sponge Hyrtios and bearing a pyrimidine ring as a spacer, exhibited in vitro cytotoxicity [32].
Since marine organisms allow the isolation of very small amount of the biologically active substances from the natural material, several total synthesis of nortopsentins were proposed [40][41][42][43]. Moreover, due to the considerable activities shown, indolyl alkaloids have attracted remarkable attention by researchers becoming an interesting field in medicinal chemistry. Thus, several dragmacidin analogues bearing six membered heterocycles, such as pyridine, pyrazine, pyrazinone and pyrimidine, as spacer and showing antiproliferative activity were synthesized [44][45][46][47].
Lately, two new series of nortopsentin analogues, 1 and 2, were efficiently synthesized and inhibited the growth of HCT-116 colorectal cancer cells at low micromolar concentrations, whereas they did not affect the viability of normal-like intestinal cells. A compound of the series 1 induced apoptosis. Instead, a derivative of the series 2 at low concentrations (GI30) induced morphological changes characteristic of autophagic death with massive formation of cytoplasmic acid vacuoles without apparent loss of nuclear material, and with arrest of cell cycle at the G1 phase, whereas higher concentrations (GI70) induced apoptosis with arrest of cell cycle at the G1 phase [57].

Biology
All the synthesized thiazoles 3a-k were evaluated by the National Cancer Institute (Bethesda MD) for cytotoxicity against the NCI-60 cell line panel using standard protocols [65]. Initially, the selected derivatives 3c,d,g,h,j,k were tested at a single dose (10 −5 M) on the full panel of approximately 60 human tumor cell lines derived from 9 human cancer cell types, that have been grouped in disease sub-panels including leukemia, non-small-cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate and breast cancers (data not shown). Compounds 3d and 3k were further selected for full evaluation at five concentration levels (10 −4 -10 −8 M).
The antitumor activity of compounds 3d and 3k was given by three parameters for each cell line: GI50 (the molar concentration of the compound that inhibits 50% net cell growth), TGI (the molar concentration of the compound leading to total inhibition of net cell growth), and LC50 (the molar concentration of the compound that induces 50% net cell death). The average values of mean graph midpoint (MG_MID) were calculated for each of these parameters.
Cell growth inhibitory activity of 3d and 3k was also investigated on human HepG2 hepatocarcinoma.
Cell growth inhibitory activity of compounds 3d and 3k was also investigated on human HepG2 hepatocarcinoma cells, a cell line not included in the NCI panel and of interest in the drug discovery because provided with a very active microsomal system for detoxification of xenobiotics. Monolayer cultures treated for 72 h with 0.1-10 μM concentrations of the compounds were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell growth. As shown in Figure 1, both the compounds inhibited the HepG2 cells growth in dose-dependent manner. Based on the dose response curve, the GI50 values were 1.69 µM) and 0.21 µM for 3d and 3k, respectively. Under identical conditions, both the nortopsentin analogues did not substantially impaired viability of normal immortalized human liver cells (Chang), suggesting high selectivity towards tumor cells.   Apoptosis induction by 3d and 3k in HepG2 cells was investigated by externalization of plasma membrane phosphatidylserine and changes of mitochondrial transmembrane potential. Flow cytometry analysis of Annexin V-FITC/PI-stained cells after 24 h treatment with the Norptopsentin analogues, assayed at their relevant GI50 values, indicated a high percentage of cells in early apoptosis, with externalized phosphatidylserine (Figure 3A).
To confirm apoptotic mechanism of cytotoxicity of the nortopsentin analogues, we carried out morphological evaluation of HepG2 cells using AO and EB double staining. After 24 h of treatment with 3d or 3k at GI50 concentration, fluorescent microscopy revealed the appearance of cells containing bright green patches in the nuclei as a consequence of chromatin condensation and nuclear fragmentation, which are typical features of apoptosis. Moreover, fluorescing orange cells owing to increase of cell permeability to ethidium bromide, cell shrinkage and nuclear fragmentation were also evident as cells in late apoptosis ( Figure 3B). Taken together, these findings provided strong evidence that the synthesized nortopsentin analogues induced apoptosis in HepG2 cells. Involvement of mitochondria in apoptosis induced in HepG2 cells by the synthesized nortopsentin analogues, was assessed. Loss of mitochondrial trans-membrane potential was indicated by decreased mitochondrial 3,30-dihexyloxacarbocyanine iodide-red fluorescence ( Figure 4A). Mitochondrial dysfunction in HepG2 cells following 24 h treatment with 3d and 3k, was also evident from the levels of intracellular ROS, revealed by cytofluorimetric analysis with 2′,7′-dichlorofluorescin diacetate, significantly higher than cell control ( Figure 4B).

General
All melting points were taken on a Büchi-Tottoly capillary apparatus. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1 H and 13 C NMR spectra were measured at 200 and 50.0 MHz, respectively, in dimethylsulfoxide (DMSO)-d6 or CDCl3 solution, using a Bruker Avance II series 200 MHz spectrometer. Compounds 3a,b were characterized only by 1 H NMR spectra because of their poor solubility. Column chromatography was performed with Merk silica gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge system). Elemental analyses (C, H, N) were within ±0.4% of theoretical values and were performed with a VARIO EL III elemental analyzer. Purity of all the tested compounds was greater than 98%, determined by HPLC as described below.

Synthesis of 2-Bromo-(1H-indol-3-yl)ethanones (9c,d)
To a cold suspension of the appropriate 3-acetylindole 8b,c (1.9 mmol) in anhydrous methanol (3.0 mL) bromine (0.1 mL, 1.9 mmol) was added dropwise. The mixture was heated at reflux for 2 h. After cooling the solvent was evaporated under reduced pressure. The residue was treated with water (20 mL), made alkaline by adding sodium hydrogen carbonate (150 mg) and extracted with EtOAc (3 × 50 mL). The organic phase was dried (Na2SO4), evaporated under reduced pressure and purified by column chromatography using DCM as eluent.

HPLC Analysis
Analysis of nortopsentin analogues was carried out using a Gilson modular liquid chromatography system (Gilson Inc., Middleton, WI, USA) equipped with M 302 and 305 pumps, and injector model 77-25 (Rheodyne, Berkeley, CA, USA) with a 20 μL injector loop and a M 802 manometric module. The chromatographic column was a µPorasil column (300 × 3.9 mm; Waters, Milford, MA, USA) provided with relevant guard cartridge (5 × 3.9 mm, Waters). Detection at 283 nm was by an M 118 UV-vis detector, used along with the Gilson 712 HPLC system controller software. Sensitivity was 0.05% absorbance unit (AUFS). Elution was with a 20 min linear gradient elution from solvent A (dichlomethane:ethyl acetate, 1:1) to 100% solvent B (ethyl acetate), at a flow rate of 1 mL/min. Retention times of 3d and 3k were 8.10 min and 8.33 min, respectively.

Viability Assay in Vitro
The tested compounds 3d and 3k were dissolved in DMSO and then diluted in culture medium so that the effective DMSO concentration did not exceed 0.1%. Human cell lines of hepatoma HepG2 and Chang liver were purchased from American Type Culture Collection, Rockville, MD, USA. Cells were grown in RPMI supplemented with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 5 μg/mL gentamicin. HepG2 culture medium also contained 1.0 mM sodium pyruvate. Cells were maintained in log phase by seeding twice a week at a density of 3 × 10 8 cells/L in humidified 5% CO2 atmosphere, at 37 °C. In all experiments, cells were made quiescent through overnight incubation before the treatment with the compounds or vehicle alone (control cells). No differences were found between cells treated with DMSO 0.1% and untreated cells in terms of cell number and viability. Cytotoxic activity of the compounds was determined by the MTT quantitative colorimetric assay based on the reduction of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) into purple formazan by mitochondrial dehydrogenases of living cells. Briefly, HepG2 and Chang cells were plated at 5 × 10 4 cells/ well in 96-well plates containing 200 μL RPMI. After an overnight incubation, cells were washed with fresh medium and incubated with the compounds in RPMI. After a 72 h incubation, cells were washed, and 50 µL FBS-free medium containing 5 mg/mL MTT were added. The medium was discarded after 4 h incubation at 37 °C, and formazan blue formed in the cells was dissolved in DMSO. The absorbance at 540 nm was measured in a microplate reader (Bio-RAD, Hercules, CA, USA). The growth inhibition activity of compounds was defined as GI50 value, which represents the log of the molar concentration of the compound that inhibits 50% cell growth.

Cell Cycle Analysis
Cell cycle stage was analyzed by flow cytometry. Aliquots of 1 × 10 6 cells were harvested by centrifugation, washed with PBS and incubated in the dark in a PBS solution containing 20 μg/mL propidium iodide (PI) and 200 μg/mL RNase, for 30 min, at room temperature. Then samples were subjected to fluorescence-activated cell sorting (FACS) analysis by Epics XL™ flow cytometer using Expo32 software (Beckman Coulter, Fullerton, CA, USA). At least 1 × 10 4 cells were analyzed for each sample.

Measurement of Phosphatidylserine Exposure
The apoptosis-induced PS externalization to the cell surface was measured by flow cytometry by double staining with Annexin V-Fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI). Annexin V binding to phosphatidylserine is used to identify the earliest stage of apoptosis. PI, which does not enter cells with intact membranes, is used to distinguish between early apoptotic cells (annexin V-FITC positive and PI negative), late apoptotic cells (annexin V-FITC/PI-double positive) or necrotic cells (annexin V-FITC negative and PI positive). HepG2 cells were seeded in triplicate in 24-wells culture plates at a density of 2.0 × 10 5 cells/cm 2 . After an overnight incubation, cells were washed with fresh medium and incubated with the compounds in RPMI. After 24 h, cells were harvested by trypsinization and adjusted at 2.0 × 10 5 cells/mL with combining buffer. One hundred μL of cell suspended solution was added to a new tube, and incubated with 5 µL annexin V and 10 μL of a 20 μg/mL PI solution at room temperature in the dark for 15 min. Then at least 1.0 × 10 4 cells were immediately subjected to FACS analysis using appropriate 2-bidimensional gating method.

Acridine Orange and Ethidium Bromide Morphological Fluorescence Dye Staining
Acridine orange (AO) stains DNA bright green, allowing visualization of the nuclear chromatin pattern and stains both live and dead cells. Ethidium bromide (EB) stains DNA orange but is excluded by viable cells. Dual staining allows separate enumeration of populations of viable non-apoptotic, viable (early) apoptotic, nonviable (late) apoptotic, and necrotic cells. Live cells appear uniformly green. Early apoptotic cells stain green and contain bright green dots in the nuclei as a consequence of chromatin condensation and nuclear fragmentation. Late apoptotic cells incorporate EB and therefore stain orange, but, in contrast to necrotic cells, the late apoptotic cells show condensed and often fragmented nuclei. Necrotic cells stain orange, but have a nuclear morphology resembling that of viable cells, with no condensed chromatin. Briefly, after HepG2 cells were treated with 3d or 3k compounds for 24 h, the medium was discarded. Cells were washed with saline 5 mM phosphate buffer, pH 7.4 (PBS) and then incubated with 100 μL PBS containing 100 μg/mL of EB plus 100 μg/mL of AO. After 20 s, EB/AO solution was discarded and cells immediately visualized by means of fluorescent microscope equipped with an automatic photomicrograph system (Leica, Wetzlar, Germany). Multiple photos were taken at randomly selected areas of the well to ensure that the data obtained are representative.

Measurement of Mitochondrial Transmembrane Potential
Mitochondrial transmembrane potential was assayed by flow cytofluorometry, using the cationic lipophilic dye 3,30-dihexyloxacarbocyanine iodide (Molecular Probes, Inc., Eugene, OR, USA), which accumulates in the mitochondrial matrix. Changes in mitochondrial membrane potential are indicated by a reduction in the 3,30-dihexyloxacarbocyanine iodide-induced fluorescence intensity. Cells were incubated with 3,30-dihexyloxacarbocyanine iodide at a 40 nmol/L final concentration, for 15 min at 37 °C. After centrifugation, cells were washed with PBS and suspended in 500 µL PBS. Fluorescent intensities were analyzed in at least 1 × 10 4 cells for each sample.

Measurement of Intracellular Reactive Oxygen Species
ROS level was monitored by measuring fluorescence changes that resulted from intracellular oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH). DCFH, at 10 mM final concentration, was added to the cell medium 30 min before the end of the treatment. The cells were collected by centrifugation for 5 min at 2000 rpm at 4 °C, washed, suspended in PBS and immediately subjected to fluorescence-activated cell sorting analysis. At least 1 × 10 4 cells were analyzed for each sample.

Conclusions
A new series of nortopsentin analogues in which the imidazole ring of the natural product was replaced by thiazole and the indole unit bound to the position 2 of thiazole was substituted by a 7-azaindole moiety was efficiently synthesized. Two of the new nortopsentin derivatives, 3d and 3k, showed good antiproliferative activity against the totality of the about 60 human tumor cell lines of NCI full panel with GI50 values ranging from low micromolar to nanomolar level (13.0-0.03 and 14.2-0.04 μM, respectively. Moreover, they have shown potent cytotoxic activity on HepG2 hepatocarcinoma cells, while under identical conditions, they did not affect normal immortalized human liver cells (Chang). Both the compounds induced a concentration-dependent accumulation of cells in the subG0/G1phase while confined viable cells in G2/M phase. The mechanism of the anti-proliferative effect of the nortopsentin derivatives was pro-apoptotic, being associated with externalization of plasma membrane phosphatidylserine and mitochondrial dysfunction.