Design, Synthesis and the Biological Evaluation of New 1,3-Thiazolidine-4-ones Based on the 4-Amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one Scaffold

New thiazolidine-4-one derivatives based on the 4-aminophenazone (4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) scaffold have been synthesized as potential anti-inflammatory drugs. The pyrazoline derivatives are known especially for their antipyretic, analgesic and anti-inflammatory effects, but recently there were synthesized new compounds with important antioxidant, antiproliferative, anticancer and antidiabetic activities. The beneficial effects of these compounds are explained by nonselective inhibition of cyclooxygenase izoenzymes, but also by their potential scavenging ability for reactive oxygen and nitrogen species. The structure of the new compounds was proved using spectroscopic methods (FR-IR, 1H-NMR, 13C-NMR, MS). The in vitro antioxidant potential of the synthesized compounds was evaluated according to the ferric reducing antioxidant power, phosphomolydenum reducing antioxidant power, DPPH and ABTS radical scavenging assays. The chemical modulation of 4-aminophenazone (6) through linkage to thiazolidine-propanoic acid derivatives 5a–l led to improved antioxidant potential, all derivatives 7a–l being more active than phenazone. The most active compounds are the derivatives 7e, and 7k, which showed the higher antioxidant effect depending on the antioxidant assay considered.

The structures of the compounds was assigned on the basis of spectral data (IR, 1 H-NMR, 13 C-NMR, MS) which are provided in the Experimental Section. In the IR spectra of ethyl 3-(2-aryl-4oxo-thiazolidin-3-yl)-propionates 4a-l the appearance of the C=O stretching band of the thiazolidine-4-one rings at 1676-1654 cm −1 , together with the characteristic C-S absorption band at 648-632 cm −1 confirm the success of the cyclization reaction and the formation of the thiazolidine system. For these compounds the characteristic absorption band of the ester group appears in the 1728-1712 cm −1 region and this band disappears in the spectra of corresponding acids 5a-l in which the characteristic carboxyl group absorption band was observed in 1743-1662 cm −1 region. The characteristic absorption band of the amide bond appears in the spectra of pyrazoline-thiazolidine-4-one derivatives 7a-l in the 1686-1652 cm −1 region.
The carboxyl group proton of the thiazolidine-propanoic acid derivatives 5a-l resonates as a singlet between 12.35-10.12 ppm. In the 1 H-NMR spectra of the pyrazoline-thiazolidine-4-one derivatives 7a-l, the amide bond proton resonates as single, doublet or multiplet between 9.67-8.96 ppm. Moreover the presence of the pyrazoline system was proved by the proton signals of two methyl groups, which resonate as singlets at 3.12-3.08 ppm and 2.33-2.16 ppm, respectively. The carbons of the pyrazoline ring appear in the 13 C-NMR spectra between 150.65-150.45 ppm and 108.38-107.83 ppm. The proton and carbon signals for other characteristic groups were all observed according to the expected chemical shift and integral values. This NMR spectral data, coupled with the corresponding mass spectra, lend strong support to the proposed structures of the all the synthesized compounds.

Ferric Reducing Antioxidant Power (FRAP) Assay
The ferric reducing antioxidant power assay is a simple and sensitive method used to evaluate the antioxidant potential of compounds. In the presence of the electron-donating compounds, the potassium ferric/ferricyanide complex is reduced to its ferrous form (Fe 2+ ) which is complexed with ferric chloride to form a blue colored complex. The amount of this complex is quantitatively determined by measuring the intensity of colour at 700 nm [24]. The reaction between the ferrous form and the ferric chloride is: The absorbance value of the samples at different concentrations (10 mg/mL, 8 mg/mL, 6 mg/mL, 4 mg/mL, 2 mg/mL in DMSO) are presented in Figure 1. The results expressed as EC 50 values (mg/mL) are shown in Table 2. Low EC 50 values indicate a higher ferric reducing antioxidant power. As we expected, the absorbance of the sample increased with the concentration, which means that reducing power of the tested compounds is concentration-dependent. The analysis of the obtained data revealed that the chemical modulation of the pyrazoline-5-one moiety through introduction of thiazolidine-4-one rings via a propioanamide chain has a great influence on antioxidant potential; all tested compounds were more active than phenazone, which was used as reference. Because phenazone showed a very low absorbance at the same concentrations as the tested compounds 7a-l, an EC 50 could not be determined for it. It was also observed that the activity of the tested compounds depends on the substituents on the thiazolidine-4-one phenyl ring. The most active compound was 7e, which has a 2-OCH 3 substituent on the phenyl ring. This compound has EC 50 = 0.122 ± 0.003, which means that it is about eight time more active than the unsubstituted compound 7a (EC 50 = 0.9647 ± 0.0108). Good activity was also shown by compounds 7d (4-Br, EC 5 = 0.4653 ± 0.0334), 7f (3-OCH 3 , EC 50 = 0.5316 ± 0.0063) and 7l (4-CH 3 ; EC 50 = 0.5455 ± 0.0177), being about twice as active as 7a. The compounds 7h (2-NO 2 ), 7i  ) and 7k (3-OCH 3 , 4-OH) also have good activity in reference to 7a. All tested compounds are less active than vitamin E at the same concentration used as positive control.  Data are mean ± SD (n = 3, p < 0.05).

Phosphomolydenum Reducing Antioxidant Power (PRAP) Assay
This assay is based on quantitative monitoring of phophomolybdenum blue complex which presents a maximum absorption band at 695 nm [25]. The absorbance value of the samples at different concentrations (1 mg/mL, 0.5 mg/mL, 0.25 mg/L, 0.125 mg/mL, 0.0625 mg/mL in DMSO) are presented in Figure 2. The results expressed as EC 50 values (mg/mL) are shown in Table 3. Low values of EC 50 demonstrate a higer phosphomolydenum reducing antioxidant power.
The data of this assay also support the conclusion that the antioxidant activity of the tested compound increases with concentration and that all tested compounds are more active than phenazone. It was observed that the presence of a 2-OCH 3 substituent on the thiazolidine-4-one phenyl ring also has a good influence on antioxidant properties, the corresponding compound 7e being the most active (EC 50 = 0.0138 ± 0.0029). In comparison with the unsubstituted compound 7a (EC 50 = 0.0153 ± 0.0010) this compound was slightly more active. The 4-CH 3 and 2-NO 2 substituents also had a good influence  on the reducing antioxidant power, as the corresponding compounds 7l (EC 50 = 00.0143 ± 0.0038) and 7h (0.0146 ± 0.0016) were also slightly more active than 7a. In this assay all the tested compounds were more active than vitamin E (0.0304 ± 0.0024) at the same concentrations used as positive control.  Data are mean ± SD (n = 3, p < 0.05).

DPPH Radical Scavenging Assay
DPPH (1,1-diphenyl-2-picrylhydrazyl) is a well-known radical which reacts with different antioxidant compounds whereby its deep violet color in methanol solution changes to yellow. The antioxidant effect is monitored by the decreasing intensity of the absorption band centered at about 515 nm [26]. The DPPH radical scavenging ability (%) of samples at different concentrations (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL in DMSO) is presented in Figure 3. Higher scavenging ability values indicate a higher radical scavenging effectiveness. The results expressed as EC 50 values (mg/mL) are shown in Table 4. Low values of EC 50 demonstrate a higher scavenging ability.   Data are mean ± SD (n = 3, p < 0.05).

The ABTS Radical Scavenging Assay
The ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay is a rapid and efficient method, based on the ability of the hydrogen donating antioxidants to scavenge the long-life radical cation ABTS + . The ABTS + is generated by the reaction between 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) and ammonium persulfate. The scavenging ability of the compounds is monitored by the decrease of the intensity of the blue colour of the ABTS + species which presents a maximum absorption band centered at about 734 nm [27].
The ABTS radical scavenging ability (%) of samples at different concentrations (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL in DMSO) is presented in Figure 4. Higher scavenging ability values indicate a higher potential radical scavenging effectiveness. The results expressed as EC 50 values (mg/mL) are shown in Table 5. Low EC 50 values indicate a higher scavenging ability.  Data are mean ± SD (n = 3, p < 0.05).
At the same concentration all tested compounds are less active than vitamin E used as positive control.

General Experimental Procedures
The melting points were measured using a Buchi Melting Point B-540 apparatus (Büchi Labortechnik AG, Postfach, Switzerland) and they are uncorrected. The FT-IR spectra were recorded on Horizon MB TM FT-IR (ABB Analytical Measurement, Québec, Canada), over a 500-4000 cm −1 range, after 32 scans at a resolution of 4 cm −1 . The spectra processing was carried out with the Horizon MB TM FTIR Software. The 1 H-NMR and 13 C-NMR spectra were obtained on a Bruker Avance400 MHz Spectrometer (Brucker, Wissemboug, France) using tetramethylsilane as internal standard and CDCl 3 as solvent, unless otherwise specified. The chemical shifts are shown in δ values (ppm). The mass spectra were registered using a Bruker MaXis Ultra-High Resolution Quadrupole Time-of-Flight Mass Spectrometer (Brucker Daltonik GmbH, Bremen, Germany). The progress of reaction was monitored on TLC, using pre-coated Kieselgel 60 F254 plates (Merck KGaA, Darmstadt, Germany) and the compounds were visualized using UV light.

Preparation of Ethyl 3-(2-Aryl-4-oxo-thiazolidin-3-yl)-propionates 4a-l
To a solution of ethyl 3-aminopropionate hydrochloride 2 (10 mmol) in freshly distilled toluene (15 mL), aromatic aldehydes (15 mmol) were added under an inert atmosphere according to the procedure described for other compounds [28]. The mixture was stirred for 5 min and mercaptoacetic acid 3 (20 mmol) was added. After 5 min, N,N-diisopropylethylamine (DIPEA, 13 mmol) was added and then the mixture was heated at 110-115 °C for 36 h until completion of the reaction (TLC monitoring, using ethyl acetate/petroleum ether, 4:6, v/v, UV light at 254 nm). The mixture was neutralized with saturated solution of sodium bicarbonate and extracted with ethyl acetate (2 × 25 mL). The organic layer was separated and washed with hydrochloric acid 1M and then with saturated solution of sodium chloride. Finally, the organic layer was dried over MgSO 4 and filtered. The solvent was removed under reduced pressure and the residue was purified on a silica gel column using ethyl acetate/petroleum ether (4:6, v/v) as eluent system.

Preparation of 3-(2-Aryl-4-oxothiazolidin-3-yl)-propanoic Acids 5a-l
To a solution of ethyl 3-(2-aryl-4-oxothiazolidin-3-yl)-propanate 4a-l (13.2 mmol) in a mixture of EtOH and THF (1:1, 25 mL:25 mL), potassium hydroxide 1 M (26 mmol) was added according to the procedure for alkaline hydrolysis of esters [30]. The mixture of reaction was stirred for 6-10 h at room temperature until completion of the reaction (TLC monitoring, using ethyl acetate/petroleum ether, 4:6, v/v, UV light at 254 nm). After that, the mixture was neutralized with hydrochloric acid 1 M to pH 2, stirred again for another 20 min and finally extracted with ethyl acetate (2 × 25 mL). The organic layer was dried over MgSO 4 and filtered. The solvent was removed under reduced pressure and the residue was triturated with ethyl ether.            [31]. The mixture was stirred for 24 h at room temperature until completion of the reaction (TLC monitoring, using dichloromethane-methanol, 10:0.5-0.8, v/v, UV light at 254). After that, the mixture was washed successively with hydrochloric acid 1M, sodium bicarbonate solution 10% and saturated solution of sodium chloride. The organic layer was collected, dried using anhydrous magnesium sulphate and concentrated by rotary evaporator. The residue was purified on a silica gel column using dichloromethane-methanol, 10:0.5-0.8, v/v as eluent system, and finally the product was triturated with cold ethyl ether.

The DPPH Radical Scavenging Assay
The antioxidant activity of the tested compounds using DPPH assay was performed in reference with [26]. For each compound different concentrations (20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL in DMSO) were tested. Briefly methanolic solution of DPPH (4 mL, 15 µM) was added to tested compounds (100 µL) in a test tube. The final concentrations of sample in the test tube were 488, 244, 122, 61, 30.5 and 15. 2µg/mL, respectively. The mixture was left for 30 min at room temperature, in the dark, and after that the absorbance was measured at 515 nm against a blank solution (methanol). The radical scavenging capacity was calculated according to the following equation: Scavenging activity % = (A control − A sample /A control ) × 100 (2) where A sample is the absorbance of the sample after 30 min. A control is the absorbance of mixture of 100 µL DMSO and 4 mL DPPH. For each compound the effective concentration (EC 50 ) was calculated by linear regression analysis and phenazone and vitamin E (α-tocopherol) were used as reference and positive control respectively. All tests were carried out in triplicate.

The ABTS Radical Scavenging Assay
The ABTS radical scavenging ability of the compounds was tested in reference with [27] with minor modifications. The ABTS + radicals were activated by reacting of ABTS (2,2'-azinobis(3ethylbenzthiazoline-6-sulphonic acid) (7 mM) with ammonium persulphate (2.45 mM) and the mixture was left at room temperature for 16 h in the dark. The ABTS + radical cation solution was diluted with ethanol to obtain an absorbance value of 0.7 ± 0.02 at 734 nm. For each compound different concentrations were tested (20, 15, 10, 5, 2.5 and 1.25 mg/mL in DMSO). To sample (50 µL), ABTS solution (1950 µL) was added. The final concentrations of sample in the test tubes were 500, 375, 250, 125, 62.5 and 31.25 µg/mL, respectively. After 6 min the absorbance was measured and the radical scavenging capacity was calculated according to the following equation: Scavenging activity % = (A t=0 − A t=6min /A t=0 ) × 100 (3) where A t=0 is the absorbance before adding the sample. A t=6 min is the absorbance after 6 min of reaction. For each sample the effective concentration (EC 50 ) was calculated by linear regression analysisand phenazone and vitamin E (α-tocopherol) were used as reference and positive control respectively. All tests were performed in triplicate.

Statistical Analysis
All antioxidant assays were carried out in triplicate. Data were analyzed by an analysis of variance (ANOVA) (p < 0.05) and were expressed as means ± SD. The EC 50 values were calculated by linear interpolation between the values registered above and below 50% activity.

Conclusions
In this study new heterocyclic compounds that combine the thiazolidine-4-one structure with pyrazoline-5-one ones have been synthesized. The structure of the new compounds was proved using spectroscopic methods (IR, 1 H-NMR, 13 C-NMR, MS). The compounds were evaluated for their antioxidant activity using in vitro assays: ferric reducing antioxidant power, phosphomolydenum reducing antioxidant power, DPPH and ABTS radical scavenging assays. The all tested compounds 7a-l showed improved antioxidant effects in reference to phenazone. The good preliminary results which support the antioxidant potential of the synthesized compounds motivate further research focused on their anti-inflammatory effects on chronic and acute inflammation models, based on implication of oxidative stress in many disorders, including inflammation.