Synthesis and in vitro antiproliferative and antibacterial activity of new thiazolidine-2,4-dione derivatives

Abstract In our present research, we synthesised new thiazolidine-2,4-diones (12–28). All the newly synthesised compounds were evaluated for antiproliferative and antibacterial activity. Antiproliferative evaluation was carried out using normal human skin fibroblasts and tumour cell lines: A549, HepG2, and MCF-7. The IC50 values were determined for tested compounds revealing antiproliferative activity. Moreover, safety index (SI) was calculated. Among all tested derivatives, the compound 18 revealed the highest antiproliferative activity against human lung, breast, and liver cancer cells. More importantly, the derivative 18 showed meaningfully lower IC50 values when compared to the reference substance, irinotecan, and relatively high SI values. Moreover, newly synthesised compounds were screened for the bacteria growth inhibition in vitro. According to our screening results, most active compound was the derivative 18 against Gram-positive bacteria. Therefore, it may be implied that the novel compound 18 appears to be a very promising agent for anticancer treatment.


Introduction
Cancer is one of the most serious health problems in the world. It is the second cause of death after heart diseases worldwide. According to the World Cancer Report 2014, 8.2 million people died from cancer in 2012 1 .
Lung, liver, stomach, and bowel cancer are the most common causes of human deaths worldwide, accounting for nearly a half of all cancer deaths. The five most common types of disease diagnosed in 2012 were lung, prostate, colorectal, stomach, and liver cancer among men; and breast, colorectal, lung, cervix, and stomach cancer among women 1 .
Despite enormous efforts aimed at the implementation of new treatment strategies of chemotherapeutic agents, treatment results in most cases are unsatisfactory 2 . Therefore, there is an urgent need to find new classes of substances with selective action against tumour cells.
Heterocyclic compounds play an important role in cancer therapy. Among them, the derivatives of thiazolidine-2,4-dione are found [3][4][5][6] . Researchers' interest in the derivatives of thiazolidine-2,4-dione has increased recently, the main reason being a wide spectrum of biological properties shown by these derivatives. It has been confirmed by numerous reviews on the activity and mechanisms of action of thiazolidine-2,4-diones 2,7,8 .
Regulation of both cell proliferation and pathways of apoptosis connected with cell death is important in understanding various diseases including malignancies 16 . Therefore, identification of regulators of the cell cycle and apoptosis stimulators is an attractive strategy to explore potential anticancer agents 17 .
There are several mechanisms of anticancer activity of thiazolidine-2,4-dione derivatives already discussed in the literature. The most known ones are induction of apoptosis, cell differentiation, and cell cycle arrest 8 . The thiazolidine-2,4-dione derivatives showing anticancer activity are mainly derivatives modified in the position 5 of the thiazolidine-2,4-dione ring.
The aim of the present research was to synthesise new thiazolidine-2,4-dione derivatives and to evaluate in vitro their potential as anticancer and antibacterial agents. As thiosemicarbazone [18][19][20] and acylhydrazone derivatives 20-24 present anticancer activity, which is similar to the activity of the above mentioned thiazolidine-2,4-diones, it was assumed that the structure modification of the thiazolidine-2,4-dione ring in position 5 by thiosemicarbazide and hydrazide derivatives can extend the biological activity of the new compounds.

Materials and methods
Melting points were determined using Fisher-Johns apparatus (Fisher Scientific, Schwerte, Germany) and were not corrected. The 1 H NMR and 13 C NMR spectra were recorded by a Bruker Avance 300 MHz instrument using DMSO-d 6 as solvent and TMS as an internal standard. Chemical shifts were expressed as d (ppm). MS using atmospheric pressure chemical ionisation (APCI) was recorded on a Bruker MicroTOF II mass spectrometer. APCI settings were as follows: vaporiser temperature, 350 C; drying gas temperature, 180 C; drying gas flow, 4 l/min; and nebuliser pressure, 2 bar. The purity of the compounds was checked by TLC on plates with silica gel Si 60 F 254 , produced by Merck Co. (Darmstadt, Germany). Elemental analyses were performed by AMZ 851 CHX analyser and the results were within ±0.4% of the theoretical value.

Antiproliferative activity in vitro
The experiment was carried out using normal human skin fibroblasts (BJ) and tumour cell lines: human lung carcinoma (A549), human hepatocellular carcinoma (HepG2), and human breast adenocarcinoma (MCF-7). All cell lines were obtained from ATCC (American Type Culture Collection, England, UK) and cultured in the appropriate, recommended by ATCC, culture medium, additionally supplemented with 10% foetal bovine serum and antibiotics: 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were maintained at 37 C in a humidified atmosphere of 5% CO 2 and 95% air. Antiproliferative activity of the new compounds was assessed by, commonly used for this purpose, colorimetric MTT assay. Cells with active mitochondrial dehydrogenases reduced intracellularly the tetrazolium dye into purple formazan, which is proportional to the number of viable cells. The cells were seeded in 96-multiwell plates in 100 ll of the appropriate complete culture medium at a concentration of 7 Â 10 3 cells/well in the case of BJ cells (normal fibroblasts), 1 Â 10 4 cells/well in the case of A549 cells (lung cancer), 1.5 Â 10 4 cells/well in the case of HepG2 cells (liver cancer), and 2 Â 10 4 cells/well in the case of MCF-7 cells (breast cancer) so as to obtain 60-70% confluent culture after 24 h of incubation at 37 Cthe cells are then in the log (logarithmic) phase, during a period of active proliferation, but still have an unoccupied space to divide. Afterwards, the culture medium was discarded and replaced with 100 ml of different concentrations of new tested compounds and reference antiproliferative agent: irinotecan. The stock solutions of the compounds were prepared in DMSO, and solvent control was being tested concurrently to exclude toxicity attributed to DMSO. Ten serial two-fold dilutions using culture medium were performed to prepare different concentrations of the tested compounds, and the highest tested concentration was 200 mg/ml. Untreated cells (maintained in the complete culture medium) served as a negative control. After exposing cells for 48 h to the tested derivatives, the MTT assay was performed as described earlier 25 . Three independent experiments (n ¼ 3) were performed in a quadruplicate. The obtained data were analysed using GraphPad Prism 5, version 5.03 software (La Jolla, CA). The IC 50 values were determined for tested compounds revealing antiproliferative activity. Moreover, safety index (SI) was calculated using the following formula: Bacteria strain susceptibility to tested agents was determined by broth microdilution method. Broth microdilution method was carried out in accordance with Clinical and Laboratory Standards Institute recommendations and was used as reference method 26 . Mueller-Hinton broth containing two-fold dilutions of the tested substances (at concentrations ranging from 3.91 to 1000 mg/ml) was used to determine their minimal inhibitory concentration (MIC) which is the lowest concentration of an antibacterial that will inhibit the visible growth of a microorganism after 18 h of incubation). Mueller-Hinton broth is recommended as the medium of choice for susceptibility testing of commonly isolated, rapidly growing aerobic, or facultative organisms. Gentamicin (KRKA, Slovenia), an aminoglycoside antibiotic active against a wide range of human bacterial infections, mostly Gram-negative bacteria including Pseudomonas, Proteus, Serratia, and the Gram-positive Staphylococcus, was used as a control antibacterial agent (at concentration from 0.12 mg to 1000 mg).
Tested compounds two-fold dilutions in Mueller-Hinton broth without bacteria were blank control wells (incubated on equal conditions). Polystyrene trays containing 96 wells were filled with serial two-fold Mueller-Hinton broth dilutions of tested agents. Next, the broth was inoculated with 20 ll of 0.5 McFarland microbial suspension (previously diluted 1:10 by Mueller-Hinton broth). Total volume per each well was 200 ll. After incubation (at 35 C for 18 h), bacterial cultures' optical density was measured by microplate reader (BioTek Synergy H4 Hybrid Reader) at 600 nm.
The MIC values were determined by the comparison of broth bacterial culture OD 600 containing tested concentrations of examined substances to blank control wells.
The structure of the new derivatives was confirmed by an elemental analysis, 1 H NMR and 13 C NMR and MS spectra. In 1 H NMR spectra, the protons of CH¼N group of all the newly synthesised compounds showed a singlet signal at d $ 8.14-8.  12.13-12.16 ppm region for compounds (12-17 and 22-26) and 12.72-12.98 ppm region for compounds as singlets [18][19][20][21]27,28 .
The presence of all carbon atoms for compounds 12-28 was confirmed by 13 C NMR spectra. For compounds 12-17 and 22-26 that are the derivatives of 2-(2,4-dioxothiazolidin-5-yl)acetic acid, carbon signal of two C¼O group of the thiazolidine ring appeared in the 172.7 and 175.8-176.0 ppm regions. Signals of two C¼O group of the thiazolidine ring for the 2-(2,4-dioxothiazolidin-5-ylilidene)acetic acid derivatives 18-21, 27, and 28 were visible at d$ 166.5-166.8 ppm and 169.3-169.6 ppm ranges, respectively. The detailed results of 1 H NMR and 13 C NMR spectra and MS are presented in the experimental part.
Molecular weight of compounds 12-21, 23, 24, and 26 were confirmed by the mass spectra. In cases of compounds 22, 25, 27, and 28, a molecular ion was not observed in the mass spectra. The fragmentation was presented in the experimental part and ways of fragmentation were depicted in the supplementary material.

Antiproliferative evaluation
Among all the new compounds tested, only 16,18,20,21,22, and 23 derivatives revealed antiproliferative activity against tumour cell lines (Table 1). Compound 16 exhibited slight anticancer activity only against human breast adenocarcinoma cells (MCF-7). However, IC 50 (concentration causing inhibition of cell proliferation by 50%) of compound 16 determined using normal human skin fibroblasts (BJ) was higher than IC 50 of compound 16 estimated using MCF-7 cells, and the SI was relatively high and >1. Note that the higher SI (SI > 1) is, the safer compound is (less cytotoxic against normal cells). Also, agents revealing SI < 1 are considered unsafe and highly cytotoxic. The slight modification of the structure of compound 16 (changing a single bond in the position 5 of the thiazolidine-2,4-dione to the double bond) resulted in a new compound 21 that revealed broader antiproliferative activity, also against human hepatocellular carcinoma cells (HepG2). However, the SI value of compound 21 was low, indicating its high cytotoxicity against normal cells. Compound 20, an analogue of the derivative 21 characterised by the lack of a methoxy group in a 4-formylphenyl, showed antiproliferative activity only against HepG2 cells. However, similar to compound 21, the derivative 20 revealed low SI value. Compound 18, characterised by the change in the configuration of formylphenyl (from 4-formyl-phenyl to 2-formyl-phenyl), appeared to be the most promising antiproliferative agent among all tested compounds. Compound 18 exhibited high antiproliferative activity against all tumour cell lines used in the experiment (lung, breast, and liver cancer) and high SI values (>1). Moreover, the derivative 18 showed meaningfully lower IC 50 values when compared to the reference agent, irinotecan, which is an inhibitor of topoisomerase I and is commonly used in cancer treatment. Compound 18 revealed the highest antiproliferative activity against human breast adenocarcinoma cells (MCF-7). In the case of MCF-7 cells, the IC 50 value of compound 18 was 13-fold lower (1.59 mg/ml) than IC 50 value of irinotecan (21.08 mg/ml), whereas the SI value of compound 18 was three-fold higher when compared to the reference substance. In the case of human hepatocellular carcinoma (HepG2) and human lung carcinoma (A549) cells, the IC 50 values of compound 18 were also very low: 3.86 mg/ml (four-fold lower compared to irinotecan) and 8.08 mg/ml (five-fold lower compared to irinotecan), respectively. Comparing the activity of compound 18 to the activity of structurally similar 2,4-dioxothiazolidine-5-acetic acid derivatives synthesised by Alegaon et al. 6 , it was observed that compound 18 showed higher activity against MCF-7 and A549 tumour cells. In their research, IC 50 of tumour cell lines MCF-7 and A549 for the most active derivative 2-(2,4-dioxo-1,3-thiazolidin-5-yl)-N-[5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-yl]acetamide was 15.28 and 12.7 mg/ml, respectively.
The derivatives of 3-formylphenyl 2-(2,4-dioxo-1,3-thiazolidin-5yl)acetate with the 4-phenyl-3-thiosemicarbazide (compound 22) and 4-(4-methylphenyl)-3-thiosemicarbazide structure (compound 23) also exhibited antiproliferative activity against all three tumour cell lines (A549, HepG2, and MCF-7), but only IC 50 of compound 23, determined using A549 cells (lung cancer), was lower compared to the reference substance. However, the derivative 23 showed antiproliferative activity against liver cancer cells (HepG2) comparable to irinotecan and with relatively high SI value (1.66), indicating that this new compound has a great potential to be used as an anticancer drug. The IC 50 values of compound 22, determined using all three cell lines, were relatively high. Nevertheless, the SI values of compound 22 were >1, indicating its high safety.
Relying on the obtained results, it may be assumed that some of the modifications introduced to the thiazolidine-2,4-dione may result in the synthesis of new compounds with a broad biological activity spectrum inter alia anticancer and antibacterial properties. Such outcome was observed especially in the case of the change in the configuration of formylphenyl (from 4-formyl-phenyl to 2formyl-phenyl) in compound 18 and introduction of the 4-(4methylphenyl)-3-thiosemicarbazide structure into compound 23. The above-mentioned compounds revealed anticancer as well as antibacterial activity. However, they showed relatively high MIC values; thus, their application just as antibacterial agents is limited. However, it should be noted that compounds 18 and 23 proved to possess very promising anticancer properties, so the extension of their activity towards antibacterial properties may be an additional advantage as cancer patients have a reduced immunity and are particularly sensitive to bacterial infections.

Conclusion
Conducted research resulted in the synthesis of new thiazolidine-2,4-dione derivatives exhibiting antiproliferative activity. Among all tested derivatives, compound 18, characterised by the change in the configuration of formylphenyl from 4-formylphenyl to 2-formylphenyl, revealed the highest antiproliferative activity against human lung, breast, and liver cancer cells. More importantly, the derivative 18 showed meaningfully lower IC 50 values when compared to the reference substance, irinotecan, and relatively high SI values. Moreover, compound 18 is characterised by antibacterial activity against some Gram-positive and Gram-negative strains. It is a very important quality as cancer patients have reduced immunity and are particularly sensitive to all kinds of infections, bacterial infections in particular. Therefore, it may be implied that the novel compound 18 appears to be a very promising agent for anticancer treatment.