DESIGN AND CYTOTOXIC ACTIVITY OF THIAZOLIDINONES VIA ONE-POT, THREE COMPONENT REACTION UNDER MICROWAVE AND TRADITIONAL METHOD

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INTRODUCTION
In recent year, chemists are interested to applied modern techniques in organic synthesis.One of these important techniques is the use of microwave irradiation for many reasons such as environmentally friendly, easy work-up, reduce time from hours to minutes, in addition to afford higher yield compared to traditional methods [1][2][3][4].Moreover, One-pot multi-component reactions are the most important application in organic synthesis due to the possibility of achieving high synthetic efficiency, exceptional synthetic efficiency, high selectivity, and procedural simplicity [1][2][3][4][5][6].4-Thiazolidinones are an important group of heterocyclic compounds, which possess a wide range of pharmaceutical and biological applications such as antiviral [10], anticancer [11,12], antimycobacterial [13], antimicrobial [14][15][16], analgesic and antiinflammatory activities [17][18][19][20].Recently, many diseases have spread for various reasons, one of the most dangerous diseases spread all over the world is cancer which caused by the excessive growth of cells in the body in an uncontrolled manner.Recently, there have been many developments in the treatment of cancer disease because cancer is the most common cause of human death according to the World Health Organization.Unfortunately, the number of patients is increasing all over the world, especially in developed countries.There are many common cancer treatments such as surgery, radiotherapy, and chemotherapy.Chemotherapy drugs is the major areas in current efforts to treat cancer [21].Therefore, the researcher team tries to find new compounds that may be later promising to treat cancer.From previous studies, we conclude that thiazole can be used in many therapeutic cases, such as cancer treatment [20,21].Therefore, the teamwork decided to use sulfamethoxazole as a starting material for the synthesis of some new compounds that may be used for treatment of cancer.This study deals with the design and synthesis of new thiazole derivatives scaffold as promising anti-cancer.
The structure of Schiff bases 3a-f were established on the basis of IR, 1 H-NMR, 13 C NMR and elemental analyses.Their IR spectra showed the absence of absorption bands due to NH2 and C=Oaldehydic groups and appearance of new absorption bands in the region 1630-1645 cm -1 due to CH=Nstr.groups. 1 H NMR (δ, DMSO-d6) spectra showed, beside the expected aromatic protons signals, new singlet signals in the region δ 8.60-8.31ppm due to N=CH ; singlet signal at δ 11.29 ppm for OH group in compound 3c; 3.83 ppm for OMe group in compound 3e; N-dimethyl group at δ 3.23 ppm and in the region δ 2.44-2.27ppm for CH3 groups in compounds 3a,b, respectively.Moreover, their 13 C NMR spectra showed the appearance of new signals at δ 55.88 ppm due to OMe group and the CH3 group at δ 21.17 ppm, respectively.Elemental analyses of compounds 3a-f provided the structure of the new compounds (cf.experimental).
IR spectra of compounds 5a-f revealed the appearance of new carbonyl groups in the region 1648-1684 cm -1 . 1 H NMR (DMSO-d6) spectra showed, beside the expected aromatic protons signals, new doublet-doublet signals in the region δ 4.03-3.47ppm consistent with the CH2 groups, and singlet signal corresponding to SCH-N groups in the region δ 6.24-6.03ppm.Furthermore, 13 C NMR spectra and elemental analyses of compounds 5a-f provided the structure of thiazolidinone ring.For example, 13 C NMR spectrum of compound 5a showed beside the expected aromatic signals the appearance of new signal at δ 21.58 ppm due to CH3 group, a new signal at δ 60.87 ppm for the CH2 group and at δ 170.11 ppm due to C=O group.Moreover, its DEPT 135 spectrum revealed the disappearance of carbonyl group and appearance of opposite side signal at δ 62.71 ppm for the CH2 group.
The formation of compounds 5a-f can be explained by the possible mechanism presented in Scheme 2.  From the recorded results in Table 1, it is clear that the low consumed time and high yield of products, when green chemistry protocols (as environment friendly and economically) have been employed and it is much better compared with conventional procedure for the synthesis of 4thiazolidinone derivatives.

Cytotoxic activity
Three compounds 5a, 5b and 5f were tested for cytotoxicity against four human tumor cells (MCF-7, HePG2, HCT 116 and 116 PC-3).Using the SRB assay after 72 hours of treatment with different concentrations (0.0, 0.01, 0.1, 1, 10, 100, and 1000 μg/mL) (cell viability assay).The findings revealed that the viability of the cells decreases as the concentration of these compounds increases in all cancer cell lines (Figure 1, Table 2).Compound 5b exhibited the most potent cytotoxic properties on HePG2 and PC-3, with IC50s of 13.7, 2.7 and 11.2, 0.3 μg/mL, respectively, followed by compound 5f on MCF-7 with an IC50 of 11.8, 1.1 μg/mL and compound 5a on PC-3 with IC50s of 13.2, 1.2 μg/mL.Furthermore, compound 5b has a promising inhabatory effect against MCF-7 and HCT 116 cells, with IC50s of 17.1, 0.62 and 16.5, 2.5 μg/mL, respectively, as well compound 5f against HePG2 and PC-3 cancer cells, with IC50s of 17.3, 2.6 and 16.1, 0.8 μg/mL, respectively.Similarly, compound 5b has a significant toxic influence on MCF-7, HePG2, and HCT 116 cells, with IC50s of 22.8, 1.9, 21.5, 2.1, and 29.1, 0.4 μg/mL, respectively, as compound 5f on HCT 116 with IC50 27.5, 1.1 μg/mL.The toxicity responding of various tested compounds to human cancer cells MCF-7, HepG2, HCT116 and PC-3., for 72 hours, cells were incubated with variable concentrations of various three compounds (Figure 1.).SRB staining was used to determine cell viability and proliferation.The amount of dye binding is directly proportional to the number of viable cells.The axes reflect the concentrations of the three test substances used in the experiment, and the percentage responses of toxicity and cell survival to the concentrations are represented by bars to observe the dose-response relationship.The extent of the decrease in cell viability can vary between cell lines and compounds, indicating differences in their sensitivity to compounds.

Chemistry
All commercially available reagents were purchased from Merck, Aldrich and Fluka, and were used without further purification.All reactions were monitored by thin layer chromatography(TLC) using precoated plates of silica gel G/UV-254 of 0.25 mm thickness (Merck 60F254) and UV light (254 nm/365 nm) for visualization.Melting points were detected with a Kofler melting points apparatus and uncorrected.Infrared spectra were recorded with a FT-IR-ALPHBROKER-Platinum-ATR spectrometer, and are given as cm -1 using the attenuated total reflection (ATR) method. 1 H NMR and 13 C NMR spectra for all compounds were recorded in DMSO-d6 on a Bruker Bio Spin AG spectrometer at 400 MHz and100 MHz, respectively.Elemental analyses were obtained on a Perkin-Elmer CHN-analyzer model.

Cell culture
The American type culture collection provided human cell lines, prostate adenocarcinoma (PC-3), breast cancer (MCF-7), hepatocellular carcinoma (HePG2) and colon cancer cell line (HCT 116) (ATCC).In a humidified, 5% (v/v) CO2 condition, cells were incubated in RPMI-1640 enriched with (100 g/mL); penicillin (100 units/L); and heat-inactivated fetal bovine serum (10 percent v/v) at 37°C.Using the sulphorhodamine B assay, the cytotoxicity of chemical compounds was assessed against human cancer cell lines (PC-3, MCF-7, HePG2 and HCT 116) (SRB).Before treated with the chemical compounds, 80 percent confluent proliferating cells trypsinized and cultivated in a 96 well tissue culture plate for 24 hours.Untreated cells (control) added to cells that exposed to the six various concentrations of each drug (0.01, 0.1, 1, 10, and 1000 µg/mL).They were exposed to the doses for 72 hours before being fixed with TCA (10% w/v) for 1 hour at 4 °C.After repeated washes, cells stained for ten min in the dark with a 0.4% (w/v) SRB solution.Glacial acetic acid, 1 percent (v/v), used to remove any remaining discoloration.The SRB-stained cells dissolved in Tris-HCl after drying overnight, and the color intensity quantified in a microplate reader at 540 nm.Using SigmaPlot 12.0 software, the correlation between viability percentage of each tumor cell line and chemical concentrations analyzed to determine the IC50 (drug dose that reduces survival to 50%) [22].

General procedure for synthesis of compounds 5a-f
Method A (traditional method).A mixture of Schiff bases 3a-f (2 mmol) and thioglycolic acid (2.2 mmol) in dry toluene and DMF (1:1, molar ratio), 30 mL) was refluxed for 7-11 h., and the water formed during the reaction was removed by Dean-Stark apparatus.After the completion of reaction (progress was checked by TLC), the reaction mixture cooled and washed with dilute solution of sodium bicarbonate to remove unreacted acid.The organic layer separated, toluene was removed by rotary evaporator, the solid product was collected and purified by crystallization from ethanol.

Method B (microwave method
).An equimolar amount (1 mmol) of sulfamethoxazole ( 1), (2 mmol), aromatic aldehyde (2 mmol) in dry toluene-DMF (5 mL) was allowed to irradiate in a MW oven for 10-14 min, then thioglycolic acid (2 mmol) was added.The mixture allowed to irradiate in a MW oven for approve time as shown in Table 1 After the completion of reaction (progress was checked by TLC), the reaction mixture was cooled and washed with dilute solution of sodium bicarbonate to remove unreacted acid.The organic layer separated, toluene was removed by rotary evaporator, the solid product was collected and purified by crystallization from ethanol.

CONCLUSION
New series of 4-thiazolidinones were synthesized via two methods, traditional method and microwave technique which is a simple and efficient protocol strategy (multicomponent reaction).The advanced of this method are mild reaction, low cost, expeditious, and an environmentally method.These compounds are promising anticancer cell and characterized by spectral techniques.The cytotoxicity of three compounds 5a, 5b and 5f were tested against four human cancer cell lines MCF-7, HePG2, HCT 116 and 116 PC-3.Compound 5b exhibited the most potent cytotoxic properties.

Figure 1 .
Figure 1.The toxicity responding of various tested compounds to human cancer cells MCF-7,HepG2, HCT116 and PC-3 using the SRB assay.

Table 1 .
Comparison of the yields and times for synthesis of 4-thiazolidinones.

Table 2 .
The IC50 (µg) of new compounds against different human solid cancer cell lines.