Thiazolidinedione or Rhodanine: A Study on Synthesis and Anticancer Activity Comparison of Novel Thiazole Derivatives.

PURPOSE
A new series of thiazolyl-2,4-thiazolidinedione / rhodanine compounds T1-T23 was synthesized and tested for their anticancer activities. Hepatocellular carcinoma cell lines were chosen due to their strong drug resistance to test the new compounds.


METHODS
All compounds were synthesized via Knoevenagel Condensation reaction and thiazolidinedione ester compounds (T3,T9,T15,T20) were hydrolyzed for obtaining the acidic compounds (T6,T12,T17,T23). All compounds were firstly screened for their anticancer activity against two hepatocellular carcinoma (HCC) cell lines, Huh7 and Plc/Prf/5 (Plc) cell lines by sulforhodamine B assay. Further IC50 values were calculated for three candidates (T4, T15, T21) in five different HCC (Huh7, Plc, Snu449, HepG2, Hep3B) and one breast cancer (Mcf7) cell line.


RESULTS
Compounds T4, T15, T21 had very strong anticancer effects even though their 10 µM concentration in Huh7 cell line. According to IC50 values, T21 was the most effective compound with IC50 values in a range from 2 to 16 µM in 6 cancer cell lines. In terms of cytotoxicity T21 mostly affected Huh7 and interestingly it was less effective against Plc.


CONCLUSIONS
Considering these results it can be suggested that compounds T4, T15 and T21 may lead to the development of more potent anticancer drugs in the future. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the deadliest type of cancers with a 5-year survival rate below 15% (1). The treatment of HCC has been a challenge so far because of its unusual resistance to chemotherapeutic agents. Sorafenib is the only drug, approved by Food and Drug Administration (FDA, United States) for targeted HCC treatment, but its effect on patient survival is minimal. So, there is a need to find novel efficient molecules acting against HCC cells.
Taking into account of the rising trend of the incidence of various organ cancers, effective therapies are urgently needed to control human malignancies. However, almost all of the chemotherapy drugs which are currently on the market cause serious side effects (2).
Thiazolidinedione (TZD) and rhodanine analog compounds have become very important groups of heterocyclic compounds in drug design and discovery. The PPAR-gamma (PPAR-γ) activating TZD medications are a class of drugs used to improve lipid and glucose metabolism in type-2 diabetes. More interestingly, numerous compounds containing the TZD ring have been developed as potential anticancer agents (3)(4). TZDs, which are anti-cancer therapeutics for the most common types of cancers including, lung, breast, and colon, have been explored for the PPAR-γ-dependent and -independent mechanisms by which TZDs exert their antitumor effects (5). Rhodanines have been reported to possess antibacterial, antifungal, antiviral, antimalarial, insecticidal, herbicidal, antitumor, anti-inflammatory and cardiotonic activities (6)(7). Besides, rhodanine derivatives are still broadly evaluated for their anticancer activity against different cancer cell lines, often exhibiting selective toxicity against normal cell lines (8)(9)(10).
Thiazoles are ubiquitous building blocks in medicinal chemistry and they can be found in numerous natural products (e.g., epothilone) and biologically important compounds including the anticancer drug dasatinib (11). On the other hand, triazoles have occupied an important role, not only in organic chemistry but also in medicinal chemistry due to their easy synthesis and attractive features as well as numerous biological activities. Furthermore, this heterocycle has a high dipole moment and it is capable of hydrogen bonding, which could be favorable in the binding of biomolecular targets. In recent years, by combining triazole with other pharmacophores, a number of compounds with potent antitumor activity were synthesized (12).
Inspired by the biological importance of these ring systems, we described the synthesis of a new series of thiazolyl-2,4-thiazolidinediones / rhodanines (T1-T23) as lead structures in developing anticancer agents (Figure 1).

Preparation of Stock Solutions
All tested drug solutions were prepared as 5 mM in DMSO and stored in -20 ºC until to use. Further dilutions of the compounds were prepared in respective media used for each cell line. DMSO was used as the control vehicle for all experiments and doxorubicin was used as positive anticancer drug control to check SRB assay efficiency.

Cancer Cell Lines
Two HCC cell lines Huh7 and Plc/Prf/5 (Plc) were used for initial screening experiments. Five hepatocellular carcinoma (HCC) cell lines [Huh7, Hep3B, Snu449, Plc and HepG2] and one breast cancer cell line Mcf7 were used for secondary screening. Cells were cultured at 37 0 C with 5% CO2 in DMEM completed with 10% FBS, 1x NEA, 2 mM L-Glutamine and 100 units penicillin/ streptomycine except Snu449 that was cultured in completed RPMI 1640.

Sulforhodamine B (SRB) Assay for Cytotoxicity
SRB assay was carried out as described elsewhere with some modifications (16). Shortly, cells were cultured in 96 well plates for 24h. After the incubation, fresh media with tested compounds were added onto the cells. When a 72h incubation period was completed, media was discarded. Cells were washed with 1xPBS once and fixed by 10% trichloroacetic acid (TCA) for 1h at 4 ºC. Overnight, dried plates were stained with 0.4% SRB in 1% acetic acid for 10 minutes at room temperature. To wash the excess of SRB, dye plates were washed with 1% acetic acid 5 times by tapping. Unbuffered 10 mM trisma base solution was added into the wells to resolubilize the SRB dye. Optic density (OD) of each well was measured by μ-Quant microplate reader in a 405 to 515 nm wave length range. Each concentration of the compounds was tested as triplicate. Average of OD values was used for calculation of cell survival percentages and IC50 values.
The structure of the synthesized compounds was elucidated by elementary analysis, 1 H NMR, 13 C NMR and mass spectral data. All spectral data were in accordance with assumed structures. It was reported that by using unsubstituted imidazolidinediones and benzaldehydes in acidic medium, the main product was the Z isomer (20). In our previous papers related with thiazolyl TZD and our other TZD compounds, all our synthesized compounds were seen in Z isomeric form (13,(21)(22)(23)(24). Besides one of our compounds (T9) was found as Z isomer (13). In this study, we used the same reaction conditions for obtaining the compounds and also, in 1 H NMR spectra, methyne protons (=CH) of the compounds T1-T23 were observed between 7.26-8.07 ppm as a singlet which correspond to the results in our recently published papers. According to our previous data, we can say that our compounds T1-T23 were formed in Z configuration. 13 C NMR data of all new compounds was reported except compounds T5, T19, T22 because of their poor solubility.
Mass analysis of compounds was performed by using ESI (+) method. All the compounds have M+H ion peaks except compounds T5 and T23. Thus, their mass analysis was performed by using ESI (-) method and their M-H ion peaks were observed.

Anticancer activity
We synthesized a new series of thiazolyl-2,4thiazolidinediones / rhodanines having pyrrolidine, morpholine, triazole ring systems for investigation of their anticancer activities. All the synthesized compounds have TZD or its isostere rhodanine ring which contains hydrogen atom, acetic acid and acetic acid ethyl ester groups on their N-3 position.
First of all, 10 µM concentrations of 23 TZD and analogue compounds were screened by sulforhodamine B assay in two HCC cell line, Huh7 and Plc (Figure 2). Three compounds (T4, T15 and T21) showed very strong cytotoxic activity against Huh7 cell line compared to Plc cell line (Table 1). In the second screening of these three compounds in 6 different cancer cell lines (5 HCC, 1 breast), it was carried out with at least 5 different concentrations (2,5 to 40µM) of the compounds. We found that T21 was the most effective anticancer agent. As it can be seen in Figure 3, IC50 values of T21 were from 5 to 16µM.
According to cell survival plots of these compounds (Figure 4a, 4b, 4c), T4 was the most tolerable agent for HCC cell lines, except Huh7 (Figure 5a). Huh7 was the most affected cell line by all three compounds and a very sharp decrease of the plotlines in Figure 4 and Figure 5a indicate that. When we compare the different cell densities at various concentrations, Plc was relatively resistant to T21 than other cell lines (Figure 4c) while Snu449 was strongly resistant to T4 and T15 ( Figure 5b). As expected, T4, T15, and T21 stopped the well-known hyper-chemosensitive Mcf7 cells (Figure 5c), however not as much as Huh7.  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T11  T12  T13  T14  T15  T17  T16  T18  T19  T20  T21  T22  T23  Growth (Figure 5c). T15 and T21 are more potent than T4 against Plc and HepG2 cell lines. In the meantime, T4 is not toxic against Snu449 while T15 and T21 have similar activity. T20 is a TZD compound and analogue of T21. Interestingly, T20 was nonresponsive compound. When their structures are compared, T20 and T21 differ only one atom from each other (O to S). This difference has destroyed completely the activity of T20. Surprisingly, none of the thiazolyl TZD or rhodanine compounds containing six membered morpholine ring have anticancer potency.
As a result, we can say that TZD and its isostere ring rhodanine can produce about similar anticancer effects for Huh7, Hep3B cell lines. Besides, it is important to declare that compounds having lipofilic acetic acid ethyl ester group instead of acetic acid or imidic hydrogen with acidic character on the N-3 position of TZD and rhodanine rings; and also, five membered heterocyclic rings such as triazole and pyrrolidine on thiazole ring have been significant for anticancer potency.

CONCLUSION
The goal of this study was synthesizing a new series of pyrrolidino / morpholino / triazolo thiazolyl-2,4-thiazolidinediones / rhodanines and then investigating their anticancer activities in hepatocellular carcinoma (HCC) cell lines. All 23 compounds were initially screened in Huh7 and Plc/Prf/5 (Plc) cell lines by sulforhodamine B assay. T4, T15, T21 were three candidates that had very strong anticancer effects even though their 10 µM concentration in Huh7 cell line, in contrast they had no effect on Plc cell line. We chose these compounds for further experiments to calculate IC50 values in five different HCC and one breast cancer cell lines. According to IC50 values, T21 was the most effective compound with IC50 values in a range from 2 to 16 µM in 6 cancer cell lines.
Compounds T4, T15 and T21 have pyrrolidine, methyl substituted triazole and phenyl substituted triazole rings, respectively. Pyrrolidino thiazole compound T4 and phenyl substituted triazolo thiazole compound T21 have rhodanine ring, while methyl substituted triazolo thiazole T15 has TZD ring. All these compounds (T4, T15 and T21) contain acetic acid ethyl ester group on N-3 position of TZD or its isostere rhodanine ring.  TZD and its isostere rhodanine compounds can produce similar anticancer effects for Huh7, Hep3B cell lines and as a conclusion, thiazole compounds bearing five membered heterocyclic rings with lipofilic group on N-3 position of TZD and rhodanine ring have been significant for anticancer potency.
The overall data presented in this study provide compounds T4, T15 and T21 as an excellent class of novel anticancer agents that may lead to the development of more potent anticancer drugs in the future.