Synthesis and Cytotoxicity Evaluation of Some Novel Thiazoles, Thiadiazoles, and Pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones Incorporating Triazole Moiety

Reactions of hydrazonoyl halides and each of methyl 2-(1-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)ethylidene)hydrazine-1-carbodithioate and 2-(1-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)ethylidene)hydrazine-1-carbothioamide afforded 2-(1-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)ethylidene)hydrazono)-3-phenyl-5-substituted-2,3-dihydro-1,3,4-thiadiazoles and 5-(4-substituted)diazenyl)-2-(2-(1-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)ethylidene)hydrazinyl)-4-arylthiazoles, respectively. Analogously, the reactions of hydrazonoyl halides with 7-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)-5-phenyl-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one gave 3-(4-substituted)-8-(5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)-6-phenyl-1-arylpyrido[2,3-d]-[1,2,4]-triazolo-[4,3-a]pyrimidin-5(1H)-ones in a good yield. The structures of the newly synthesized were elucidated via elemental analysis, spectral data and alternative synthesis routes whenever possible. Twelve of the newly synthesized compounds have been evaluated for their antitumor activity against human breast carcinoma (MCF-7) and human hepatocellular carcinoma (HepG2) cell lines. Their structure activity relationships (SAR) were also studied. The 1,3,4-thiadiazole derivative 9b (IC50 = 2.94 µM) has promising antitumor activity against the human hepatocellular carcinoma cell line and the thiazole derivative 12a has promising inhibitory activity against both the human hepatocellular carcinoma cell line and the breast carcinoma cell line (IC50 = 1.19, and 3.4 µM, respectively).


Cytotoxic Activity
Our literature survey showed that many thiazole and 1,3,4-thiadiazole derivatives have antitumor activity with excellent IG50 and IC50 values, as depicted in Figure 1 [55][56][57][58]. In view of these facts, we examined the antitumor activity of a new series of substituted thiadiazoles and thiazoles against the human breast carcinoma cell line (MCF-7) and against the human hepatocellular carcinoma (HepG2).  The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drug doxorubicin using a crystal violet colorimetric viability assay. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of the cell population (IC50) was determined. The cytotoxic activity was expressed as the mean IC50 of three independent experiments (Table 1) and the results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner. The small values of IC50 for the selected compounds indicate that, for more anticancer effect higher concentrations can be used. The results are represented in Table 1, Figures 2 and 3 show that: -The in vitro inhibitory activities of tested compounds against the human breast carcinoma (MCF-7) have the following descending order: The in vitro inhibitory activities of tested compounds against the human hepatocellular carcinoma (HepG2) cell line have the following descending order:   -For substituents at position 2 of the 1,3,4-thiadiazole ring, the in vitro inhibitory activity of tested compounds against the human breast carcinoma cell line have the following descending order: CONHC6H5 > COOC2H5 > C6H5 > C6H5CO > C10H7CO > CH3CO > C4H3SCO group. -For substituents at position 2 of the 1,3,4-thiadiazole ring, the in vitro inhibitory activity of tested compounds against the human hepatocellular carcinoma cell line have the following descending order: COOC2H5 > C6H5 > C6H5CO > CONHC6H5 > C10H7CO > CH3CO > C4H3SCO group.

General
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus. IR spectra were recorded in potassium bromide discs on PyeUnicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. 13 C-NMR was recorded on a Bruker spectrometer at 75 MHz. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyses were measured by using an ElementarVario LIII CHNS analyzer. Antitumor activity of the productswas carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. Hydrazonoyl halides 4 [59][60][61][62][63][64][65] were prepared as reported in the respective literature.
To a mixture of alkyl carbodithioate 3 (0.305 g, 1 mmol) and the appropriate hydrazonoyl halide 4a-g (1 mmol) in ethanol (20 mL), triethylamine (0.5 mL) was added, the mixture was stirred at room temperature for 2 h. The resulting solid was collected and recrystallized from dimethylformamide to give the corresponding 1,3,4-thiadiazolines 9a-g. The products 9a-g together with their physical constants are listed below.

Alternate Synthesis of 12g
To a solution of 14 (0.374 g, 1 mmol) in ethanol (20 mL) was added sodium acetate trihydrate (0.138 g, 1 mmol), and the mixture was cooled to 0-5 °C in an ice bath. To the resulting cold solution was added portionwise a cold solution of benzenediazonium chloride [prepared by diazotizing aniline (1 mmol) dissolved in hydrochloric acid (6 M, 1 mL) with a solution of sodium nitrite (0.07 g, 1 mmol) in water (2 mL). After complete addition of the diazonium salt, the reaction mixture was stirred for a further 30 min in an ice bath. The solid that separated was filtered off, washed with water and finally recrystallized from DMF to give a 76% of a product which was identical in all aspects (m.p., mixed m.p. and IR spectra) with those obtained from reaction of 11 with 4d.

Evaluation of the Antitumor Activity Using Viability Assay
Human breast carcinoma (MCF-7) and human hepatocellular carcinoma (HepG2) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and were subcultured two to three times a week.
Potential cytotoxicity of the compounds was evaluated on tumor cells using the method of Gangadevi and Muthumary [66]. The cells were grown as monolayers in growth RPMI-1640. The monolayers of 10 4 cells adhered at the bottom of the wells in a 96-well microtiter plate incubated for 24 h at 37 °C in a humidified incubator with 5% CO2. The mono layers were then washed with sterile phosphate buffered saline (0.01 M, pH 7.2) and simultaneously the cells were treated with 100 µL from different dilutions of tested sample in fresh maintenance medium and incubated at 37 °C. A control of untreated cells was made in the absence of tested sample. Positive control containing doxroubcin drug was also tested as reference drug for comparison. Six wells were used for each concentration of the test sample. Every 24 h the observation under the inverted microscope was made. The number of the surviving cells was determined by staining the cells with crystal violet [66,67] followed by cell lysing using 33% glacial acetic acid and reading the absorbance at 590 nm using a microplate reader (SunRise, TECAN, Inc, Männedorf, Switzerland) after mixing well. The absorbance values from untreated cells were considered as 100% proliferation. The number of viable cells was determined using the microplate reader as previously mentioned and the percentage of viability was calculated as [1 − (ODt/ODc)] × 100%, where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from the graphic plots.

Conclusions
Some newly synthesized compounds were evaluated for their anti-cancer activity against the human breast carcinoma (MCF-7) and human hepatocellular carcinoma (HepG2) cell lines. Also, their structure activity (SAR) was studied. The results revealed that the thiazole derivative 12a has promising antitumor activities (IC50 = 3.41 and 1.12 µM, respectively) and most of the tested compounds showed moderate anti-cancer activities.

Author Contributions
AOA, SMG designed research; AOA, SMG and SAA performed research, analyzed the data, wrote the paper. AOA, SMG read and approved the final manuscript.