Synthesis and Antitumor Activity of New Thiazole Nortopsentin Analogs

New thiazole nortopsentin analogs in which one of the two indole units was replaced by a naphthyl and/or 7-azaindolyl portion, were conveniently synthesized. Among these, three derivatives showed good antiproliferative activity, in particular against MCF7 cell line, with GI50 values in the micromolar range. Their cytotoxic effect on MCF7 cells was further investigated in order to elucidate their mode of action. Results showed that the three compounds act as pro-apoptotic agents inducing a clear shift of viable cells towards early apoptosis, while not exerting necrotic effects. They also caused cell cycle perturbation with significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population.


Introduction
In the latest decades, marine environment has increasingly provided a huge number of biologically active molecules. Among marine organisms, deep-sea sponges have contributed with several compounds endowed with antitumor activity [1][2][3][4]. The isolation of such molecules is very important since cancer is still an important social problem, in fact it is supposed to maintain as causes of death its primacy after heart and circulatory disorders. This scenario justifies the attention paid by a multitude of researchers in the individuation and development of natural or synthetic heterocyclic compounds as scaffold for antitumor agents [5][6][7][8]. Bis-indolyl alkaloids represent an important class of deep-sea sponge metabolites, useful as leads for antitumor agents. They are characterized by two indole units linked, through their position 3, by a spacer [9,10]. The spacer can be an acyclic chain such as in hyrtiosin B, isolated from Hyrtios erecta [11], or a carbocyclic ring as in the case of asterriquinone, isolated from Aspergillus fungi [12]. Heterocyclic rings can also play as spacer for bis-indolyl alkaloids. Thus, dragmacidin isolated from the deep water sponges Dragmacidon, Halicortex bears a saturated six-membered piperazine ring (Chart 1) [13].
The interesting results obtained by the aza-substitution of the indole moiety, led to the synthesis and biological evaluation of 3-[2-(1H-indol-3-yl)-1,3-thiazol-4-yl]-1H-4-azaindoles and the corresponding 1H-7-azaindole derivatives (Chart 2) [27,28]. Both series showed potent antiproliferative activity against a wide range of cell lines, including diffuse malignant peritoneal mesothelioma (DMPM), a fatal disease, poorly responsive to conventional therapies, and acted as CDK1 inhibitors. Moreover, a derivative belonging to the 7-aza series, in the mouse model, by intraperitoneal administration was effective in a significant reduction of the DMPM at well tolerated doses. Lately, three new series of nortopsentin analogs of type 1, 2 and 3 (Chart 2) were efficiently synthesized and exhibited remarkable antiproliferative activity against several human tumor cell lines [29,30].
Interestingly, a derivative of the series 2 at low concentrations (GI30) caused morphological changes typical 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 [29].
Considering the interesting biological activity of nortopsentin analogs and in particular of 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindole derivatives previously reported by us [26], herein we report the synthesis of new derivatives of type 4, 5 (Scheme 1) and 6 (Scheme 2), in which one of the two indole units was replaced by a naphthyl portion, to further investigate the contribution of the aryl moiety on biological activity. The antiproliferative activity of the novel compounds was evaluated in different human cancer cell lines and further studies were performed on the most active derivatives, in order to clarify their mechanism of action.
Interestingly, a derivative of the series 2 at low concentrations (GI 30 ) caused morphological changes typical 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 (GI 70 ) induced apoptosis with arrest of cell cycle at the G1 phase [29].
Considering the interesting biological activity of nortopsentin analogs and in particular of 3-(2-phenyl-1,3-thiazol-4-yl)-1H-7-azaindole derivatives previously reported by us [26], herein we report the synthesis of new derivatives of type 4, 5 (Scheme 1) and 6 (Scheme 2), in which one of the two indole units was replaced by a naphthyl portion, to further investigate the contribution of the aryl moiety on biological activity. The antiproliferative activity of the novel compounds was evaluated in different human cancer cell lines and further studies were performed on the most active derivatives, in order to clarify their mechanism of action.
3-[4-(Naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6a-h were synthesized (Table 2), also in this case, by Hantzsch reaction between the key intermediates indolo-3-carbothiamides 22d, 23a-d, 24a-c and naphthalene-2-acetylbromide 25, performed in dimethylformamide (DMF) under reflux (Scheme 2). In particular, reaction of naphthalene-2-acetylbromide 25 with N-Boc indolo-3-carbothiamides 24a-c afforded the corresponding unprotected 3-[4-(naphthalene-2-yl)-1,3-thiazol-2-yl]-1H-indoles 6e-h. Indolo-3-carbothiamides 22d, 23a-d and 24a-c were prepared from the corresponding indoles 8a-d, 9a-d and 18a-c through the formation of amides 19d, 20a-d and 21a-c as previously reported by us [28].    Table 3 are shown the growth percentages calculated for some of the nortopsentin analogs since those derivatives for which growth percentages higher than 90 were measured against all the three lines are not reported. Compounds 4a, 6a and 6d appeared the most active compounds in inhibiting cell growth and their activity was further investigated on MCF-7 cells, which are the most sensitive to the cytotoxic property of the compounds. When assayed in the concentration range 0.1-100 µM, they inhibited the growth of MCF-7 cells in dose-dependent manner ( Figure 1) and on the basis of GI 50 value, the drug concentration effective in causing 50% inhibition of cell growth, compound 4a appeared the most effective (Table 4).   Table 3 are shown the growth percentages calculated for some of the nortopsentin analogs since those derivatives for which growth percentages higher than 90 were measured against all the three lines are not reported. Compounds 4a, 6a and 6d appeared the most active compounds in inhibiting cell growth and their activity was further investigated on MCF-7 cells, which are the most sensitive to the cytotoxic property of the compounds. When assayed in the concentration range 0.1-100 μM, they inhibited the growth of MCF-7 cells in dose-dependent manner ( Figure 1) and on the basis of GI50 value, the drug concentration effective in causing 50% inhibition of cell growth, compound 4a appeared the most effective (Table 4).

Cell Death Mechanism
The mechanism of the most active compounds, 4a, 6a and 6d, in inducing cell death (necrosis or apoptosis) was investigated by double staining with propidium iodide (PI) and Annexin V-FITC followed by cytofluorimetric analysis. As shown in Figure 2, all three compounds induced a clear shift of viable cells towards early apoptosis in MCF-7 cells after 24 h treatment, while did not exert necrotic effects.

Cell Death Mechanism
The mechanism of the most active compounds, 4a, 6a and 6d, in inducing cell death (necrosis or apoptosis) was investigated by double staining with propidium iodide (PI) and Annexin V-FITC followed by cytofluorimetric analysis. As shown in Figure 2, all three compounds induced a clear shift of viable cells towards early apoptosis in MCF-7 cells after 24 h treatment, while did not exert necrotic effects.

Cell Cycle Analysis
The distribution of MCF-7 cells in the cell cycle phases after 24 h treatment with the three compounds 4a, 6a and 6d, was assessed by flow cytometric analysis after staining of DNA with PI. All synthesized compounds caused a significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population ( Figure 3).

Cell Cycle Analysis
The distribution of MCF-7 cells in the cell cycle phases after 24 h treatment with the three compounds 4a, 6a and 6d, was assessed by flow cytometric analysis after staining of DNA with PI. All synthesized compounds caused a significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population (Figure 3).

Cell Death Mechanism
The mechanism of the most active compounds, 4a, 6a and 6d, in inducing cell death (necrosis or apoptosis) was investigated by double staining with propidium iodide (PI) and Annexin V-FITC followed by cytofluorimetric analysis. As shown in Figure 2, all three compounds induced a clear shift of viable cells towards early apoptosis in MCF-7 cells after 24 h treatment, while did not exert necrotic effects.

Cell Cycle Analysis
The distribution of MCF-7 cells in the cell cycle phases after 24 h treatment with the three compounds 4a, 6a and 6d, was assessed by flow cytometric analysis after staining of DNA with PI. All synthesized compounds caused a significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population (Figure 3).

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)-d 6 solution, using a Bruker Avance II series 200 MHz spectrometer. Compounds 5c,d 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).

Viability Assay In Vitro
Cytotoxic activity of the compounds against human tumor cell lines was determined by the MTT colorimetric assay based on the reduction of 3-(4,5-dimethyl-2-thiazolyl)bromide-2,5-diphenyl-2H-tetrazolium to purple formazan by mitochondrial dehydrogenases of living cells. This method is commonly used to illustrate inhibition of cellular proliferation. Monolayer cultures were treated with various concentrations (0.1-100 µM) of the drugs. Briefly, all cell lines were seeded at 2 × 10 4 cells/well in 96-well plates containing 200 µL RPMI. When appropriated, cells were washed with fresh medium and incubated with the compounds in RPMI. After 72 h incubation, cells were washed, and 50 µL FBS-free medium containing 5 mg/mL MTT were added. The medium was discarded after 2 h incubation at 37 • C by centrifugation, and formazan blue formed in the cells was dissolved in DMSO. The absorbance, measured at 570 nm in a microplate reader (Bio-RAD, Hercules, CA, USA), of MTT formazan of control cells was taken as 100% of viability. The growth inhibition activity of compounds was defined as GI 50 value which represents the log of the molar concentration of the compound that inhibits 50% cell growth. Each experiment was repeated at least three times in triplicate to obtain the mean values.

Cell Cycle Analysis
Cell cycle stage was analyzed by flow cytometry. MCF-7 cells (5.0 × 10 4 cells/cm 2 ) were seeded in triplicate in 24-wells culture plates. After an overnight incubation, the cells were washed with fresh medium and incubated with the compounds or vehicle alone (control cells) in RPMI for 24 h. Then cells were harvested by trypsinization. Aliquots of 1 × 10 6 cells were 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 of at least 1.0 × 10 4 cells were subjected to FACS analysis.

Conclusions
New thiazole nortopsentin analogs in which one of the two indole units was replaced by a naphthalyl portion were conveniently synthesized. Among these, compounds 4a, 6a and 6d showed good antiproliferative activity in particular against MCF7 cell line with GI 50 values in the micromolar range. Biological studies performed to clarify their mechanism of action showed that the three compounds act as pro-apoptotic agents inducing a clear shift of viable cells towards early apoptosis in MCF-7 cells after 24 h treatment, while not exerting necrotic effects. They also caused cell cycle perturbation with significant decrease in the percentage of cells in the G0/G1 and S phases, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population.