The Synthesis and Antiproliferative Activities of New Arylidene-Hydrazinyl-Thiazole Derivatives

New and known arylidene-hydrazinyl-thiazole derivatives have been synthesized by a convenient Hantzsch condensation. All compounds were evaluated for their in vitro cytotoxicity on two carcinoma cell lines, MDA-MB231 and HeLa. Significant antiproliferative activity for 2-(2-benzyliden-hydrazinyl)-4-methylthiazole on both MDA-MB-231 (IC50: 3.92 µg/mL) and HeLa (IC50: 11.4 µg/mL) cell lines, and for 2-[2-(4-methoxybenzylidene) hydrazinyl]-4-phenylthiazole on HeLa (IC50: 11.1 µg/mL) cell line is reported. Electrophoresis experiments showed no plasmid DNA (pTZ57R) cleavage in the presence of the investigated thiazoles.

Regarding the synthesis of hydrazinyl-thiazoles, two procedures have been highlighted in the literature: the classical condensation of a carbonyl group with thiosemicarbazide followed by the cyclization of thiosemicarbazones with α-halocarbonyl derivatives [12][13][14][15], and a more recently reported one-step multi-component synthetic protocol [16,17].
The main goal of this work was to identify new possible chemotherapeutic agents based on organic heterocyclic derivatives, which are less harmful for the human body than the well-known platinum derivatives. In this paper, we present a two-step protocol for the synthesis of seven new arylidene-hydrazinyl-thiazoles 2c, 2f, 2h, 2j, 2l, 2m, 2p and nine previously reported thiazoles 2a, 2b, 2d, 2e, 2g, 2i, 2k, 2n, 2o, followed by the in vitro evaluation of the antiproliferative activity on two carcinoma cell lines, MDA-MB231 and HeLa. To identify a possible correlation between DNA damage and cytotoxicity, the interaction of the thiazole derivatives 2a, 2e, 2h, 2i with DNA was evaluated by electrophoresis.

Synthesis of Arylidene-Hydrazinyl-Thiazole Derivatives 2a-p
A series of arylidene-hydrazinyl-thiazole derivatives 2a-p were synthesized in two steps: the condensation of aromatic aldehydes with hydrazinecarbothioamide, followed by the cyclization of aryliden-thiosemicarbazones 1a-e with α-halocarbonyl derivatives (Scheme 1, Table 1). Both the condensation and cyclization reactions were performed in good yield by the Hantzsch protocol. Derivatives 2a, 2b, 2d, 2e, 2g, 2i, 2k, 2n and 2o have been previously prepared by other groups [17][18][19][20].  NMR and MS spectra were recorded for all the arylidene-hydrazinyl-thiazoles 2a-p. The 1 H-NMR spectra of arylidene-hydrazinyl-thiazoles 2a-p present a similar pattern for the hydrazone unit. The most downfield singlet, around 12 ppm, corresponds to the hydrazinyl moiety (N-NH), which is only present in DMSO-d6 solutions, and otherwise missing due to the deuterium exchange. The singlet around 8.4~7.8 ppm is assigned to the azomethine proton (CH=N). The expected molecular ion (M + ) is found in the mass spectra of all arylidene-hydrazinyl-thiazoles 2a-p. Moreover, the fragmentation pathway involved the cleavage of the nitrogen-nitrogen bond from the hydrazinyl unit. For example, in the MS spectra of thiazole 2a, this fragmentation generates a peak at m/z 113 for the aza-thiazole ion, while for the thiazole 2b the corresponding aza-thiazole ion peak is observed at m/z 175, in accordance with the substitution of the thiazole heterocycle.

In Vitro Cytotoxicity Assay
The anti-proliferative activity of the sixteen arylidene-hydrazinyl-thiazole derivatives against two human carcinoma MDA-MB231 and HeLa cell lines was evaluated using MTT assays [14,21] after 24 h of treatment. According to the IC50 data (Table 2), five thiazole derivatives, 2a, 2e, 2f, 2h and 2i, have shown significant inhibition on both MDA-MB231 and HeLa cancer cell lines. Their activity is comparable or even better than that of the platinum drugs cisplatin and oxaliplatin, which were used as controls.
Having the IC50 values for thiazoles 2a-p, we tried to establish a correlation between the cytotoxic activity and the molecular structure, by looking at the nature of the functional groups and their position on the arylidene-hydrazinyl-thiazole backbone. The presence of a methyl or phenyl group in position 4 (see Scheme 1) and a hydrogen or acetyl in position 5 on the thiazole ring, combined with phenyl, p-OH-phenyl or p-MeO-phenyl as the aromatic group attached to the hydrazinyl unit, led to compounds 2a, 2e, 2f, 2h and 2i, which exhibited the highest antiproliferative activity. On the other hand, the presence of chlorine atoms at the phenyl hydrazinyl unit and ethyl carboxylate group in position 5 on the thiazole ring 2m-o decreased the antiproliferative efficiency (Table 2).  The viability of the breast cancer MDA-MB-231 cells and cervical cancer HeLa cells decreased with an increase in the concentration of the thiazole derivatives 2a-p (Figures 1 and 2). The profiles of the MDA-MB-231 cells survival viability, correlated to the thiazole doses, revealed a common trend for thiazoles 2a, 2f, 2i, as well as cisplatin and oxaliplatin ( Figure 1). For the HeLa cell line, the same correlation is observed between compounds 2a, 2e, 2h, 2i and the chemotherapeutic drugs cisplatin and oxaliplatin ( Figure 2).

DNA Intercalation Study
For the best candidates, the arylidene-hydrazine-thiazole derivatives 2a, 2e, 2h and 2i, their interactions with plasmid DNA (pTZ57R) were investigated. Gel electrophoresis experiments investigating the plasmid migration in agarose gel after incubation with thiazoles 2a, 2e, 2h and 2i did not reveal any ability of these compounds to generate changes in the electrophoretic mobility of supercoiled DNA. These electrophoresis experiments showed that DNA cleavage does not occur in the presence of thiazoles 2a, 2e, 2h and 2i, even at elevated concentrations of the selected compounds ( Figure 3).
The gel electrophoresis results for the pTZ57R DNA incubated with thiazole derivatives 2a, 2e, 2h and 2i suggested that, despite increasing thiazole concentrations, no meaningful effect on plasmid DNA was observed. This is an indication that the cytotoxic effect of these derivatives against MDA-MB-231 and HeLa cell lines does not involve interaction with DNA.

Materials and Methods
The starting materials and solvents were obtained from commercial sources. The reagents used for cell culture experiments (fetal calf serum (FCS), penicillin-streptomycin, glutamine and RPMI 1460 cell media) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antineoplastic drugs cisplatin and oxaliplatin were purchased from Actavis Sindan Pharma (Bucharest, Romania). The GeneRuler 1 kb DNA ladder was purchased from Thermo Scientific (Waltham, MA, USA).
Compounds 1a-e were prepared according to the literature [14]. Melting points were measured with an Electrothermal IA 9200 apparatus (Bibby Scientific Limited (Group HQ), Stone, UK), and are uncorrected values. 1 H-NMR and 13 C-NMR spectra were recorded in CDCl3, acetone-d6 or DMSO-d6 (locked to Me4Si) using a 400 or 600 MHz Bruker Avance NMR spectrometer (Bruker Biospin GmbH, Rheinsberg, Germany). Elemental analysis was carried out on a Vario EL III instrument. The mass spectra were recorded with a Shimadzu QP 2010 Plus GC-MS instrument (Shimadzu Corporation, Kyoto, Japan) and a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).
The equipment involved in cell lines multiplication included Class II LaminAir laminar hoods, a ShelLab incubator, and an Eppendorf Centrifuge 5702R with spin-out rotor. Spectrophotometric measurements were completed with Biotek Synergy 2 Multi-Mode Microplate Reader with SQ Xenon Flash light source, using well-area colorimetric scanning.
The MTT experimental data were processed with Graph Pad Prism 5 biostatistics software (Sheldon Manufacturing, Inc., Cornelius, OR, USA).
For the cytotoxicity assessment, two highly proliferative MDA-MB-231 and HeLa tumor cells were utilized. Both cell lines used in the experiment were purchased from the European Collection of Cell Cultures (ECCAC, BioTek, Winooski, VT, USA).

General Procedure for the Synthesis of Compounds 2a-p
A mixture of arylidene-hydrazine-carbothioamide (10 mmol) and α-halogenocarbonyl derivative (10 mmol) in acetone/DMF (10 mL, v/v: 1/0.2) was stirred at room temperature for 20-24 h; the reaction progress was monitored by TLC (toluene/AcOEt 2/1 v/v; silica plates). The reaction mixture was neutralized at pH 7 with NaHCO3 aqueous solution (10%). The precipitate was filtered and recrystallized from ethanol or acetic acid. For all compounds the yield, the melting point, the EI MS, and the elemental analysis are given, while the 1 H-and 13 C-NMR data are only provided for the new derivatives.

Cell Cultures
Both MDA-MB-231 and HeLa cell lines were grown under sterile conditions in Cole-type culture flasks (25 cm 2 , Nunclon Easy Flask), using cell growth media (RPMI 1460) supplemented with 5% fetal calf serum (FCS), 0.1% penicillin-streptomycin, and 0.1% glutamine. The culture flasks were kept in an incubator at constant humidified atmosphere, temperature (37 °C), and CO2 level (5%). The cells passage was performed by enzymatic methods using Trypsin.

Cell Treatment and Cytotoxicity Evaluation
For cytotoxicity evaluation, the stock solutions of thiazoles 2a-p in DMSO (2000 μg/mL) were used to prepare diluted samples with the following concentrations: 0.1, 1, 6.25, 12.50, 25 and 50 µg/mL using RPMI 1460 media. The cells were placed on flat bottom 96-well micro plates for tissue culture (ca. 10.000 cells/well) and cultured in complete medium as described above (200 μL).
After 24 h, the solutions of thiazoles 2a-p were added separately to each well. After treatment (24 h) with thiazole derivatives, the culture medium was removed from the wells, without disturbing the attached cells, and 60 μL of MTT-Hanks media solution was added to each well. After incubating the plates for 2 h at 37 °C, the MTT solution was removed, and the formazan crystals were solubilized by adding DMSO (100 μL).
The 96-well plates were measured with a multimode microplate reader, by monochromator-based absorbance detection at 570 nm wavelength. The optical density, quantified by colorimetric measurements, is directly proportional with the amount of formazan crystals formed in the cells and it is an indicator of the cellular viability.
Untreated cells were used as reference for cell proliferation. For each compound, reagent blank (media and MTT) and color control (wells containing media and thiazole derivatives solution, without cells) were used. For Positive control, two antineoplastic drugs, cisplatin and oxaliplatin, were used in the same concentrations as the studied compounds. All experiments were performed in triplicate.

DNA Electrophoresis Tests
The plasmid DNA was purified from an overnight culture of Escherichia coli DH5α cells using the DNA-spin™ Plasmid DNA Purification Kit (ThermoScientific).

Conclusions
A series of arylidene-hydrazinyl-thiazole derivatives 2a-p were synthesized with good yields by the Hantzsch protocol and their structures confirmed by NMR spectroscopy and mass spectrometry. The in vitro cytotoxicity was evaluated for all thiazoles 2a-p on two carcinoma cell lines, MDA-MB231 and HELA. An excellent inhibition of cancer cells proliferation was reported for five thiazole derivatives. Among them, 2-(2-benzyliden-hydrazinyl)-4-methylthiazole 2a exhibited a significant antiproliferative activity on both MDA-MB-231 (IC50: 3.92 µg/mL) and HeLa (IC50: 11.4 µg/mL) cell lines, while 2-[2-(4-methoxybenzylidene) hydrazinyl]-4-phenylthiazole 2e showed a similar cytotoxic effect (IC50 value 11.1 µg/mL) on the HeLa cell line. It was also shown that these thiazole derivatives do not interact with plasmid DNA.