Design, synthesis and evaluation of anticancer activity of novel 2-thioxoimidazolidin-4-one derivatives bearing pyrazole, triazole and benzoxazole moieties

A novel series of substituted 2-thiohydantoin incorporated with benzoimidazole, pyrazole, triazole and/or benzoxazole moieties has been synthesized using (E)-3-[1-(4-bromophenyl)ethylideneamino]-2-thioxoimidazolidin-4-one 1 as the key starting material. The key material 1 also, reacted with an acetic anhydride, aromatic aldehydes, secondary amines, formaldehyde and triethyl orthoformate to give the corresponding acetyl, chalcone, Mannich bases and ethoxymethylene derivatives, respectively. The structures of the novel compounds were confirmed by spectral data and elemental analysis. The cytotoxic activity of all synthesized compounds was assessed in vitro against human hepatocellular cancer cell line (HePG-2) and breast carcinoma cell line (MCF-7). The bioassay results revealed that compound 14 has the best activity against HePG-2 cell line (IC50 = 2.33 μg/mL), while compound 5 has the best activity against MCF-7 cell line (IC50 = 3.98 μg/mL). Electronic supplementary material The online version of this article (10.1186/s13065-018-0418-1) contains supplementary material, which is available to authorized users.

To obtain substituted 2-thiohydantoin derivatives incorporated with benzoimidazole and/or benzoxazole moieties, compound 1 was reacted with triethyl orthoformate and/or diethyl oxalate in xylene in the presence of sodium metal under reflux to give 12 and/or 13, respectively. Compound 13 was condensed with o-phenylenediamine and/or 2-aminophenol in acetic acid under fusion to give 14 and/or 15, respectively (Scheme 4). Morover, new series of biologically active 2-thiohydantoin derivatives were prepared by acetylation of 1 with acetic anhydride to give 16 and 17. Condensation of 16 with aldehydes such as vanillin in the presence of piperidine under fusion gave chalcone derivative 18. Also, Mannich base was prepared by reacting 1 with diethylamine and formaldehyde in ethanol to give 19. Finally, hydrazinolysis of 1 with hydrazine hydrate in ethanol gave 20 (Scheme 5). The structures of the synthesized compounds were confirmed by spectral data and elemental analysis.

Biological assessment In vitro anticancer screening
The anti-tumor activity of all synthesized compounds has been evaluated against two cell lines HepG-2 cells (human hepatocellular cancer cell line), and MCF-7 (breast carcinoma cell line) [34][35][36]. The cell lines were obtained from VACSERA Tissue Culture Unit, and the experiments were performed by the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Different concentrations of the tested samples (500, 250, 125, 62.5, 31.25, 15.6, 7.8, 3.9, 2 and 1 µg/mL) were used to detect the inhibitory activity. Cell viability (%) was determined by colorimetric method. Doxorubicin was used as the reference drug, as it is one of the most effective anticancer agents. The relationship between drug concentration and cell viability was plotted to obtain the survival curve of hepatocellular carcinoma cell line HePG2 and breast cancer cell line MCF-7. The IC 50 value, which corresponds to the concentration required for 50% inhibition of cell viability was calculated.
Tables 1, 2, 3 show the in vitro cytotoxicity of the synthesized compounds against hepatocellular carcinoma cell line HePG-2. Tables 4, 5, 6 show the in vitro cytotoxicity against breast carcinoma cell line MCF-7. Data examination revealed that the tested compounds showed good to moderate activity. Compound 14 has the best activity against HePG-2 cell line (IC 50 = 2.33 μg/mL), while, compound 5 has the best activity against MCF-7 cell line (IC 50 = 3.98 μg/mL) and compound 11a has the lowest activity against HePG-2 cell line (IC 50 = 243 μg/ mL) and against MCF-7 cell line (IC 50 = 249 μg/mL). The structure and biological activity relationship of the title compound 1 showed that, the activity of thiohydantoin diverse with the substituents on it, where introducing active groups such as, CH 3 CO, OH, OCH 3 , OC 2 H 5 , =CH-OC 2 H 5 enhanced the activity, also, the presence of benzoimidazole, pyrazolone, pyrazole carbonitrile and triazine moieties enhanced the activity of thiohydantoin, while the activity was decreased by introducing benzoxazole, pyrazolidinedione moieties and Schiff bases. Alkylation of thiohydantoin decreases the activity; however, when the ester 2 was reacted with hydrazine hydrate to form the acid hydrazide 4 the activity was enhanced especially against MCF-7 cell line.
The resulting data of the 50% inhibition concentration (IC 50 ) summarized in Table 7 showed that, the synthesized compounds have different activity against hepatocellular carcinoma cell line HePG2 and breast cancer cell line MCF-7.

Conclusion
A novel series of substituted 2-thiohydantoin with incorporated benzoimidazole, pyrazole, triazole and/ or benzoxazole moieties has been synthesized. The structures of these compounds were confirmed by IR, 1 HNMR, 13 CNMR, MS and elemental analysis. The bioassay results revealed that, Compound 14 has the best activity against HePG2 cell line (IC 50 = 2.33 μg/mL), while compound 5 has the best activity against MCF-7 cell line (IC 50 = 3.98 μg/mL). Structure and biological activity relationship showed that, the activity of thiohydantoin diverse with the substituents on it, where introducing active groups such as, CH 3 CO, OH, OCH 3 , OC 2 H 5 , =CH-OC 2 H 5 and the presence of benzoimidazole, pyrazolone, pyrazole carbonitrile and triazine moieties enhanced the activity of thiohydantoin.

Experimental section
Melting points were measured using electrothermal digital melting points apparatus and are uncorrected. IR (infrared) spectra were recorded on NICOLET (iS50 FT-IR) spectrometer using KBr pellets. 1 H and 13 C NMR (nuclear magnetic resonance) were recorded on a Bruker AS 850 TM spectrometer at 850 MHz and chemical shifts were given with respect to TMS (tetramethylsilane). Mass (MS) spectra were recorded on GC/MS with CI (chemical ionization) and a Hewlett-Packard MS Engine Thermospray and ionization by electron impact to (70 eV). Microanalysis was conducted using elemental analyzer 106.

Typical procedure for syntheses of compounds 5-7
A mixture of compound 4 (0.01 mol) and an equimolar amount of ethyl acetoacetate or acetylacetone or ethyl cyanoacetate (or diethyl malonate) was refluxed in 10 mL of acetic acid for 5 h. The product formed after cooling was filtered off, washed with water, dried and crystallized with acetic acid to give compounds 5, 6, and 7, respectively.

Synthesis of (E)-5-amino-1-(2-{3-[1-(4-bromophenyl) ethylideneamino]-4-oxo-2-thioxoimidazolidin-1-yl} acetyl)-1H-pyrazole-4-carbonitrile 8
To compound 4 (0.01 mol) dissolved in 50 mL absolute ethanol was added slowly with shaking, ethoxymethylenemalononitrile (0.01 mol), after addition of about half of the quantity, the solution was carefully heated to boiling. The remaining ethoxymethylenemalononitrile was added, at such a rate to maintain gentle boiling of the solution, after all the ethoxymethylenemalononitrile had been added, the solution was gently boiled for an additional 30 min and finally was set aside overnight in the refrigerator. The product formed was filtered off, dried and crystallized from ethanol to give 8 as yellow crystals, in yield 84%, m.p 198-200 °C. 1

Syntheses of compounds 11a and 11b
A mixture of 4 (0.01 mol), aromatic aldehydes such as, (isonicotinaldehyde and anisaldehyde) (0.01 mol) and piperidine (1 mL) was fused on a hot plate at 100-110 °C for half an hour, then ethanol (25 mL) was added and refluxed for 2 h. The reaction mixture then cooled and acidified with diluted hydrochloric acid. The resulting solid was filtered off, washed with water, dried and purified by crystallization from proper solvent to give 11a, b.

Syntheses of compounds 12 and 13
A mixture of 1 (0.01 mol) and triethyl orthoformate and/ or diethyl oxalate (0.01 mol) in xylene (25 mL) in the presence of sodium metal (0.50 g), was heated under reflux for 4 h, then filtered upon hot and the filtrate then concentrated, cooled and the solid formed was filtered off, dried and purified by crystallization from ethanol to give 12 and 13, respectively.

Syntheses of compounds 14 and 15
A mixture of 13 (0.01 mol) and o-phenylenediamine or 2-aminophenol (0.01 mol) in acetic acid (25 mL) was fused under reflux for 2-3 h, then cooled. The solid formed was filtered off, washed with ethanol, dried and purified by crystallization from ethanol to give 14 and 15.

Cytotoxicity Assay
In 96-well plate, the cells were seeded at a cell concentration of 1 × 10 4 cells per well in 100 µL of growth medium. Different concentrations from the tested sample in fresh medium were added after 24 h of seeding. The tested compounds underwent serial two-fold dilutions, then added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. For a period of 48 h, the microtiter plates were incubated in a humidified incubator with 5% CO 2 at 37 °C. For each concentration of the samples, three wells were used. Control cells were incubated without test sample and with or without DMSO. After incubation of the cells at 37 °C, various concentrations of the sample were added, and the incubation was continued for 24 h and viable cells yield was determined by a colorimetric method.
In brief, media were aspirated and the crystal violet solution (1%) was added to each well for at least 30 min after the end of the incubation period. All excess stain is removed, where the plates were rinsed using tap water. To all wells, glacial acetic acid (30%) was then added and mixed thoroughly, and then the absorbance of the plates was measured after gently shaken on the Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated. The optical density was measured with the microplate reader (Sunrise, TECAN, Inc, USA) to determine the number of viable cells 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 (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve for each conc. using Graphpad Prism software (San Diego, CA. USA).