Microwave Assisted Synthesis of Some New Fused 1,2,4-Triazines Bearing Thiophene Moieties With Expected Pharmacological Activity

Rapid and efficient solvent-free synthesis of 4-amino-3-mercapto-6-[2-(2-thienyl)vinyl]-1,2,4-triazin-5(4H)-one 1 under microwave irradiation is described. Some new fused heterobicyclic nitrogen systems such as 1,2,4-triazino[3,4-b][1,3,4]thiadiazinones, 1,3,4-thiadiazolo[2,3-c][1,2,4]triazinone and pyrazolo[5,1-c]-[1,2,4]triazine-7-carbonitrile, have been synthesized by treatment of 1 with bifunctional oxygen and halogen compounds, CS2/KOH and malononitrile via heterocyclization reactions, in addition to some uncondensed triazines. Structures of the products have been deduced from their elemental analysis and spectral data (IR, 1H-NMR, 13C-NMR). Select new synthesized compounds were screened as anticancer agents, with some showing activity as cytotoxic agents against different cancer cell lines.


Introduction
Microwave assisted organic synthesis (MAOS) continues to affect synthetic chemistry significantly by enabling rapid, reproducible and scaleable chemistry development [1][2][3][4][5]. The use of microwave irradiation is an established tool in organic synthesis for achieving better selectivity, rate enhancement and reduction of thermal degradation byproducts [6,7]. Moreover it is an acknowledged quick alternative and green synthetic organic chemistry technology that also typically results in easier work-up procedures. However these procedures are practically limited as under the high temperatures produced in a microwave oven solvents create high pressure, which may cause explosions. One of the ways to overcome this problem is the use of organic reagents on solid inorganic supports, which has attracted attention because of enhanced selectivity, milder reaction conditions and associated ease of manipulation [8,9]. It also provides an opportunity to work with open vessels and enhances the option of scaling up reactions [10,11].
In light of this we planned to synthesize a series of new 1,2,4-triazines carrying thiophene moieties in the hope of obtaining new products of superior biological activity such as anticancer activity.
Hoping to expand the biological activity, compound 1 was next condensed with aromatic aldehydes such as benzaldehyde or 2-thiophenaldehyde in EtOH-HCl to give the Schiff base products 12 a and 12 b , respectively (Scheme 3). The structures of 12 a and 12 b were confirmed from their spectral data. Thus, the 1 H-NMR recorded the disappearance of the NH 2 peak of 1 and the appearance of new peaks due to the benzene and thiophene rings. The reaction of 1 with maleic anhydride under microwave irradiation yielded the N-(2,5-dioxopyrrolyl)-1,2,4-triazine derivative 13 (Scheme 3). The structure of 13 was confirmed from its IR, 1 H-NMR, 13 C-NMR and elemental analysis. The IR showed a broad band at 1675-1667 cm −1 due to three amide C=O groups, while the 1 H-NMR showed a peak at δ = 7.58 ppm for the pyrrole protons. The structure of compound 15 was confirmed from its elemental analysis and its spectral data as well. The 1 H-NMR recorded a broad signal at δ = 3.95 ppm due to NH 2 protons and at 4.00 ppm for SCHCO, while its 13 C-NMR spectrum showed a signal at δ = 38.2 ppm for SCHCO.

Cytotoxicity of the Compounds against Hep-G2 Cells
Using the MTT assay we studied the effect of the compounds on the viability of cells after 48 h incubation. Incubation of Hep-G2 cell line with gradually increasing doses of all the compounds led to insignificant changes in the growth of Hep-G2 cells, as indicated from their IC 50 values (>20 µg/mL), except for compounds 4, 7, 11, 12b and 15, which showed inhibition in the viability of Hep-G2 cells compared with the growth of untreated control cells, as concluded from their low IC 50 values, as indicated by black bars in Figure 1. The positive control, paclitaxol, which is a known anti-cancer drug, resulted in high cytotoxicity against Hep-G2 cells with an IC 50 value of 643 ng/mL ( Figure 1, Table 1).

Cytotoxicity of the Compounds against MCF-7 Cells
Using the MTT assay we studied the effect of the compounds on the viability of MCF-7 cells after 48 h incubation. Incubation of cell line with most of the tested compounds led to insignificant changes in the growth of MCF-7 cells as indicated from their IC 50 values (>20 µg/mL), except for compounds 7 and 12b, which possessed an inhibitory effect on MCF-7 cells viability, compared with the growth of untreated control cells, as concluded from their low IC 50 values, indicated by black bars in Figure 2. The positive control, paclitaxol, which is a known anti-cancer drug, resulted in high cytotoxicity against MCF-7 cells with an IC 50 value of 452 ng/mL ( Figure 2, Table 1).

Cytotoxicity of the Compounds against HCT-116 Cells
The effect of the compounds on the viability of HCT-116 cells after 48 h incubation was studied by the MTT assay. Incubation of HCT-116 cell line with gradually increasing doses of some tested compounds led to insignificant changes in the growth of HCT-116 cells, as indicated from their IC 50 values (>20 µg/mL). On the other hand, compounds 4, 7, 11, 12b, and 15 gave a significant inhibition in the viability of HCT-116 cells, compared with the growth of untreated control cells, as concluded from their low IC 50 values, as iindicated by black bars in Figure 3. The positive control, paclitaxol, which is a known anti-cancer drug, resulted in high cytotoxicity against HCT-116 cells with an IC 50 value of 709 ng/mL ( Figure 3, Table 1).

Percentage of Induced Apoptotic and Necrotic Cells in Hep-G2 Cells
According to the findings of the cytotoxicity experiments, compounds 4, 7 and 12b possessed a potent cytotoxic effect against Hep-G2 cells. To detect the type of cell death induced in the cells by those compounds, Hep-G2 cells were treated with the IC 50 values of each compound for 6 h and the apoptosis and necrosis cell population percentages was recorded using acridine orange/ethidium bromide staining. As shown in Figure 4, all of the tested compounds led to an apoptosis-dependant cell death (66-91% of the total dead cell number), while the percentage of necrotic cells was only 9-34% of the total dead cell number, except for compound 15, which mainly induced necrotic cell death up to 64% ( Figure 4, Table 2).

Percentage of Induced Apoptotic and Necrotic Cells in MCF-7 Cells
According to the cytotoxicity experiment results, compounds 7 and 15 possessed a potent cytotoxic effect against MCF-7 cells. To detect the type of cell death induced in the cells by those compounds, MCF-7 cells were treated with the IC 50 values of each compound for 6 h and the apoptosis and necrosis cell population percentages was recorded using acridine orange/ethidium bromide staining. As shown in Figure 5, both of the tested compounds led mainly to an apoptosis-dependant cell death (64-72% of the total dead cell number), while the percentage of necrotic cells were only 28-36% of the total dead cell number ( Figure 5, Table 3).

Percentage of Induced Apoptotic and Necrotic Cells in HCT-116 Cells
According to the findings of the cytotoxicity experiments, compounds 4, 7, 11, 12b, and 15 possessed a potent cytotoxic effect against HCT-116 cells. To detect the type of cell death induced in the cells by those compounds, HCT-116 cells were treated with the IC 50 values of each compound for 6 h and the apoptosis and necrosis cell population percentages was recorded using acridine orange/ethidium bromide staining. As shown in Figure 6, the tested compounds 4, and 7 resulted in an apoptosis-dependant cell death (61-84% of the total dead cell number), while compounds 11 and 15 resulted in necrosis-dependant cell death (59-71% of the total dead cell number). On the other hand compound 12b induced both cell death types ( Figure 6, Table 4).

Cell Culture
Several human cell lines were used in testing the anti-cancer activity including: hepatocellular carcinoma (Hep-G2), colon carcinoma (HCT-116), and histiocytic lymphoma and breast adenocarcinoma (MCF-7) (ATCC, VA, USA). HCT-116 cells were grown in Mc Coy's medium, while all cells were routinely cultured in DMEM (Dulbeco's Modified Eagle's Medium) at 37 °C in humidified air containing 5% CO 2 . Media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, containing 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulphate, and 250 µg/mL amphotericin B. Monolayer cells were harvested by trypsin/EDTA treatment, while and leukemia cells were harvested by centrifugation. Compound dilutions were tested before assays for endotoxin using Pyrogent ® Ultra gel clot assay, and they were found endotoxin free. All experiments were repeated four times, unless mentioned, and the data was represented as (mean ± S.D.). Cell culture material was obtained from Cambrex BioScience (Copenhagen, Denmark), and all chemicals were from Sigma (NY, USA).

Cytotoxicity Assay
Cytotoxicity of tested samples against different types of cells was measured using the MTT Cell Viability Assay. The MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and form a dark blue insoluble formazan crystals which is largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is directly proportional to the level of soluble formazan dark blue color. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm [50].

Procedure
Cells (0.5 × 10 5 cells/well) in serum-free media were plated in a flat bottom 96-well microplate, and treated with 20 µL of different concentrations of each tested compound for 48 h at 37 °C, in a humidified 5% CO 2 atmosphere. After incubation, media were removed and 40 µL MTT solution/well were added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 µL of acidified isopropanol/well and plate was shaken at room temperature, followed by photometric determination of the absorbance at 570 nm using microplate ELISA reader. Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells compared with the vehicle control, with cytotoxicity indicated by <100% relative viability.

Calculations
Percentages of relative viability were calculated using the following equation:

[Absorbance of treated cells / Absorbance of control cells] × 100
Then the half maximal inhibitory concentration IC 50 was calculated from the equation of the dose response curve.

Apoptosis and Necrosis Staining
The type of cell death was investigated in compound-treated and untreated cells using acridine orange/ethidium bromide staining [51,52]. In brief, cells were treated with the IC 50 value of each promising compound for 6 h and collected to be treated with acridine orange/ethidium bromide mixture. The vital, necrotic, and apoptotic cells were counted. A mixture of 100 µg/mL acridine orange and 100 µg/mL ethidium bromide was prepared in PBS. The cell uptake of the stain was monitored under a fluorescence microscope, and the apoptotic, necrotic, and viable cells were counted. The early apoptotic cells had yellow chromatin in nuclei that were highly condensed or fragmented. Apoptotic cells also exhibited membrane blebbing. The late apoptotic cells had orange chromatin with nuclei that were highly condensed and fragmented. The necrotic cells had bright orange chromatin in round nuclei. Only cells with yellow, condensed, or fragmented nuclei were counted as apoptotic cells in a blinded, nonbiased manner.

Conclusions
Taken together, this work revealed that compounds 4, 7, 12b and 15 may be active cytotoxic agents against different cancer cell lines. This cytotoxic effect was found to be mainly due to apoptosis, which indicated that those compounds may be promising candidate anti-cancer agents, subject to further study. From the chemistry point of view, cytotoxic effect may be due to the presence of free thiol or thioether groups in these compounds.

General
All melting points were taken on an Electrothermal IA 9100 series digital melting point apparatus. The IR spectra (KBr) discs were recorded on a Perkin-Elmer 1650 spectrometer. 1 H-and 13 C-NMR spectra were recorded on a Bruker AC-300 Hz instrument. Chemical shifts were expressed as δ (ppm) relative to TMS as internal standard and DMSO-d 6 as solvent. The elemental analysis were performed at the Micro-analytical Center, Cairo University. Mass spectra were recorded on a Shimadzu GC-MS-QP 1000 EX spectrometer. A domestic microwave oven was used (2450MHz, 800W). The pharmacological study was carried out at the National Research Center (Center of Excellence for Advanced Sciences, Cancer Biology Research Laboratory). All chemicals were from Sigma (NY, USA). (1). Method A: a mixture of 2-oxo-4-(2-thienyl)but-3-enoic acid (0.01 mol) and thiocarbohydrazide (0.01 mol) in glacial acetic acid (25 mL) was stirred under reflux for 2 h, cooled to room temperature, and the precipitate that separated was collected by filtration to give yellowish crystals (yield 62%), m.p. 252-255 °C. Method B: a mixture of 2-oxo-4-(2-thienyl)but-3-enoic acid (0.01 mol) and thiocarbohydrazide (0.01 mol), were dissolved in a mixture of methylene chloride/methanol (80/20, 15 mL) then silica gel (1.0 g, 200-400 mesh) was added, the solvent was removed by evaporation, and the dried residue was transferred into a glass beaker and irradiated for 1.  (2). A mixture of 2-oxo-4-(2-thienyl)but-3-enoic acid (0.01 mol) and thiocarbohydrazide (0.01 mol) was dissolved in a mixture of methylene chloride/methanol (80/20, 15 mL) then silica gel (1.0 g, 200-400 mesh) was added, the solvent was removed by evaporation, the dried residue was transferred into a glass beaker and drops of glacial acetic acid were added then the mixture was irradiated for 1.

General Procedure for Preparation of 12 a and 12 b
To a solution of 1 (0.01 mol) in thanol (20 mL) the appropriate aldehyde (0.01 mol) was added followed by HCl (1 mL) and the reaction mixture was refluxed for 2 h, the reaction left to cool to room temperature then poured onto crushed ice and neutralized with dil. ammonium hydroxide. The precipitate formed was collected and crystallized from ethanol (20 mL). (12 a (13). A mixture of 1 (0.01 mol) and maleic anhydride (0.01 mol), was dissolved in a mixture of methylene chloride/methanol (80/20, 15 mL) then silica gel (1.0 g, 200-400 mesh) was added, the solvent was removed by evaporation. The dried residue was transferred into a glass beaker and irradiated for 1.5-2.0 min in a domestic microwave oven (2450 MHz, 800 W). The product was chromatographed on a silica gel column, using methylene chloride as eluent. Yield 66%, m.p. 330-333 °C. IR (KBr): broad band at 1675-1667 cm −1 (3C=O amide). 1