Synthesis and Anticancer Activity of Some New S-Glycosyl and S-Alkyl 1,2,4-Triazinone Derivatives

A series of S-glycosyl and S-alkyl derivatives of 4-amino-3-mercapto-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (1)were synthesized using different halo compounds such as preacetylated sugar bromide, 4-bromobutylacetate, 2-acetoxyethoxy-methyl bromide, 3-chloropropanol, 1,3-dichloro-2-propanol, epichlorohydrin, allyl bromide, propargyl bromide, phthalic and succinic acids in POCl3. The structures of the synthesized compounds have been deduced from their elemental analysis and spectral (IR, 1H-NMR, and 13C-NMR) data. Some of the synthesized compounds were screened as anticancer agents. Significant anticancer activities were observed in vitro for some members of the series, and compounds 4-Amino-3-(3-hydroxypropylthio)-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (12) and 3-(4-Oxo-3-(2-(2-thienyl)vinyl)-4H-[1,3,4]thiadiazolo-[2,3-c][1,2,4]tr-iazin-7-yl)propanoic acid (18) are active cytotoxic agents against different cancer cell lines.


Results and Discussion
For the above reasons we sought to synthesize a series of S-glycosyl and S-alkyl of 4-amino-3mercapto-6-(2-(2-thienyl)vinyl)-1,2,4-triazin-5(4H)-one (1) [32] which should combine all the above benefits in one target and to then test the anticancer activity of these synthesized compounds against a panel of human cell lines including hepatocellular carcinoma (Hep-G2), colon carcinoma (HCT-116), and histiocytic lymphoma and breast adenocarcinoma (MCF-7) (ATCC, VA, USA). In our previous work we prepared compound 1 by two different methods [33]. The synthetic routes to S-glycosides 3-7 via microwave irradiation (MWI, 2,450 MHz, 800 W) and conventional methods are illustrated in Scheme 1. The appearance of signals at δ 1.87, 1.92, 1.95, 2.02 ppm due to the four CH 3 of four OAc groups and at δ 6.21 ppm due to the anomeric proton of sugar with J = 9.60 Hz in the 1 H-NMR confirms the formation of the β-configuration of S-glucoside 3. This was also confirmed by the IR spectrum which showed a bands at 3,310, 1,739 and 1,660 cm −1 for NH 2 and two ester and amide C=O groups.
The 13 C-NMR spectrum of 3 showed signals at δ 61.63, 66.24, 68.47, 72.88, 73.13 ppm characteristic for sp 3 carbon in a sugar moiety, in addition to δ 86.73 ppm for the anomeric carbon (C 1 ). In compound 6 the IR spectrum showed a band at 1,737 cm −1 for the acetoxy groups of the sugar and the 1 H-NMR displayed a signal at δ 6.46 ppm with J = 2.80 Hz characteristic for the anomeric protons, in addition, in the 13 C-NMR spectrum the anomeric carbon appear at δ 87.6 ppm.
The deacetylated compounds 4 and 7 showed in the IR spectra the disappearance of the acetoxy groups and the appearance of bands at 3,434 and 3,421 cm −1 for the resulting free hydroxyl groups. The 1 H-NMR spectra and elemental analysis confirmed the structure as shown in the Experimental section. When compound 1 was treated with alkylating agents such as 4-bromobutylacetate, 2-acetoxyethoxymethyl bromide [34], 3-chloropropanol, 1,3-dichloro-2-propanol and epichlorohydrin using DMF as a solvent in the presence of K 2 CO 3 , acetyloxy S-alkyl 1,2,4-triazinones 8, 10 and S-alkyl 1,2,4-triazinones 12-14 were obtained (Scheme 2). Deacetylation of compounds 8 and 10 in the presence of TEA/MeOH and few drops of water [35][36][37] yielded the deacetylated S-alkyl 1,2,4triazinone 9 and 11, respectively (Scheme 2). The structures of compounds 8 and 10 were confirmed by the presence of the (two) C=O bands in the IR spectrum at 1,735 and 1,969 cm −1 for the acetoxy and amide groups. Compounds 9, 10, 12 and 13 showed characteristic OH group bands at 3,450-3,486 cm −1 . In addition, the 1 H-NMR spectrum of 8 showed signals at δ 1.91 and 4.06 ppm characteristic for CH 3 CO and SCH 2 groups, respectively, while compound 10 showed the deshielded signal at δ 4.45 ppm for the SCH 2 O group.

Scheme 2. Synthesis of some
The 1 H-NMR spectrum of compound 12 gave signals at δ 1.98, 3.53 and 4.48 ppm characteristic for CH 2(g) , CH 2(f) and CH 2(h) groups, in addition to a triplet signal at δ 4.85 ppm for the OH group which exchanged with D 2 O. Its 13 C-NMR showed signals at δ 30.30, 50.08, 58.02 ppm characteristic for CH 2(g) , CH 2(f) and CH 2(h) , respectively., while the 1 H-NMR spectrum of 14 showed signals at δ 4.05-4.18, 4.25 ppm for the CH 2 -O and CH-O of an epoxypropyl moiety, in addition to signals at δ 4.67 and 4.87 ppm for the diastereotropic protons of the S-CH 2 grouping. IR, 1 H-NMR and elemental analysis data for compounds 9, 11 and 13 were in agreement with the assigned structures as shown in the Experimental section. Alkylation of compound 1 with allyl and propargyl bromide in the presence of K 2 CO 3 /acetone afforded S-alkyl triazine derivatives 15 and 16 (Scheme 3).  The 1 H-NMR spectrum of 15 showed doublet signals at δ 5.06, 5.24 and 5.31 ppm characteristic for SCH 2 and the terminal olefin =CH 2 protons, while its 13 C-NMR spectrum gave a signal at δ 60.1 ppm for SCH 2 carbon. Menawhile, the 13 C-NMR for compound 16 showed signals at δ 48.3 and 76.6, 77.1 ppm for SCH 2 and C≡C carbons.
In the reaction of compound 1 with phthalic and succinic acids in the presence of POCl 3 , an intramolecular cyclization took place giving the thiadiazolo[2,3-c] [1,2,4]triazine derivatives 17 and 18, respectively (Scheme 3). The structures of compound 17 and 18 were supported by elemental analysis and spectral data. The IR spectra of 17 and 18 showed the absence of the (NH 2 ) bands and the presence of C=O and OH bands of acids in the 1,710-1,715 and 3,445-3,463 cm −1 range. In addition, the 1 H-NMR spectra of these compounds revealed the presence of signals at δ 12.18 and 12.28 ppm for the OH group of an acid, while in compound 18 there are two triplet signals at δ 1.82 and 2.68 ppm for the CH 2 -CH 2 protons, respectively. In addition the 13 C-NMR spectrum showed signals at δ 26.80 and 32.50 ppm for CH 2 -CH 2 carbon and eleven sp 2 signals for the aromatic carbons, three C=N and two C=O groups.

Cytotoxicity of the compounds against MCF-7 Cells
Using the MTT assay we studied the effect of the compounds on the viability of cells after 48 h incubation. Incubation of MCF-7 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 12 and 18, 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 IC 50 value of 452 ng/mL (Table 1, Figure 2).

Cytotoxicity of the compounds against HCT-116 Cells
The effect of the compounds on the viability of cells after 48 h incubation was studied by MTT assay. Incubation of HCT-116 cell line with gradual doses of some tested compounds led to insignificant change in the growth of HCT-116 cells as indicated from their IC 50 values (>20 µg/mL). On the other hand, the compounds 12, 15 and 18 resulted in 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 indicated 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 IC 50 value of 709 ng/mL (Table 1, Figure 3).

Percentage of induced apoptotic and necrotic cells in Hep-G2 Cells
According to the cytotoxicity experiments, compounds 12 and 18 possessed potent cytotoxic effects 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, both of the tested compounds led to an apoptosis-dependant cell death (84-86% of the total dead cell number), while the percentage of necrotic cells were only 14-16% of the total dead cell number (Table 2, Figure 4).

Percentage of induced apoptotic and necrotic cells in MCF-7 Cells
According to the findings of the cytotoxicity experiment, compounds 12 and 18 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 to an apoptosis-dependant cell death (85-92% of the total dead cell number), while the percentage of necrotic cells were only 8-15% of the total dead cell number (Table 3, Figure 5).  apoptosis-dependant cell death (61-94% of the total dead cell number), while the percentage of necrotic cells were only 6-39% of the total dead cell number (Table 4, Figure 6).  Figure 6. The type of cell death was investigated in HCT-116 cells after the treatment with the promising cytotoxic compounds, using acridine orange/ethidium bromide staining to compare between the percentage of necrotic cells (grey segment) and the apoptotic cells (black segment). Data are representing mean value ± SE.

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-, 13 C-NMR spectra were determined on Bruker AC-300 MHz instrument. Chemical shifts are expressed as δ (ppm) relative to TMS as internal standard and DMSO-d 6 as solvent. The elemental analysis and mass spectra were carried by 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 (2,450 MHz, 800 W). The pharmacological studies were carried out in National Research Center (Center of Excellence for Advanced Sciences, Cancer Biology Research Laboratory). All chemicals were from Sigma.

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 ng/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 (USA).

Cytotoxicity assay
Cytotoxicity of tested samples against different types of cells was measured using the MTT Cell Viability Assay. 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 [38].

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 shacked 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
Percentage of relative viability was 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 [39,40]. 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/ethedium 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.

General method for preparation of compounds 3 and 6
A mixture of compound 1 (10 mmol) and peracetylated sugar (10 mmol) was dissolved in methylene chloride (20 mL), then silica gel (1 g, 200-400 mesh) was added, the solvent was removed by evaporation and the dried residue was irradiated for 5 min in a domestic microwave oven (2,450 MHz, 800 W). The product was extracted with methylene chloride, evaporated to dryness and purified by recrystallization from ethanol. -3-(2`,3`,4`,6`-tetra-O-acetyl-β-D-glucopyranosylthio)-6-[2-(2-thienyl) (30 mL) was added to a solution of compound 1 (10 mmol) in aqueous (distilled water, 10 mL) potassium hydroxide (10 mmol). The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, the residue was washed with distilled water to remove the inorganic residue and then the formed solid product dried and crystallized from ethanol. Yield 25%; m.p. 85-87 °C.

Conclusions
In this work we prepared the S-glycosyl and S-alkyl derivatives of 1,2,4-triazinone, in addition to thiadiazolotriazines. From the anticancer tested compounds its revealed that compounds 12 and 18 are active cytotoxic agents against different cancer cell lines, to a variable extent. This cytotoxic effect was found to be mainly due to apoptosis, which indicated that those compounds may act as promising candidate anticancer agents. From the chemistry point of view, the cytotoxic effects may be due to the presence of free CH 2 CH 2 CH 2 OH or CH 2 CH 2 COOH groups in these compounds.