Synthesis and Antioxidant Activity of Novel Thiazole and Thiazolidinone Derivatives with Phenolic Fragments

: In this work, a series of thiosemicarbazones with phenol fragments were used as starting compounds for the synthesis of new effective antioxidants containing both a phenol substituent and a heterocyclic fragment: thiazole and thiazolidinone. To determine the most stable conformation of thiosemicarbazone, a potential energy scan was used, along with NOESY NMR spectroscopy data. A number of thiazole derivatives were obtained due the interaction of thiosemicarbazones with several bromoketones: bromoacetophenone, bromodimedone, and bromoacetylcoumarin. The product yields varied from 71 to 94%. Thiazolidinone derivatives were obtained through the reaction be-tween thiosemicarbazones and chloroacetic acid or maleic anhydride with good yields of 82–95%. The antioxidant activities of all the products were determined in vitro: the radical cation scavenging activity was estimated using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), while the ferric reducing capacity was determined using the ferricyanide/Prussian blue method. It was found that the antioxidant activity of most synthesized substances in both tests exceeds the activity of 4-methyl-2,6-di-tert -butylphenol, while derivatives with a fragment of 2,6-di-tert -butylphenol have the highest activity.


Introduction
The formation of reactive oxygen species (ROS), including free radicals and peroxides of both inorganic and organic origin, is a result of normal cells functions.An overproduction of ROS causes redox imbalance of the cell's homeostasis and triggers oxidative stress.This state is very harmful for the cell, since ROS are responsible for the oxidation of cellular macromolecules such as carbohydrates, lipids, proteins, and DNA [1].Oxidative stress is the cause or an important component of many severe diseases, including hypertension [2], atherosclerosis [3], Alzheimer's disease [4], diabetes [5], and infertility [6], and is also one of the components of chronic fatigue syndrome [7].
The mechanism of antioxidants' action in different systems has been studied for several decades, but no universal approach for the development of new antioxidant molecules has been identified yet.The most up-to-date rational design approach for effective antioxidants is to combine in one molecule fragments that interfere with the action of various oxidizing agents.For example, phenols can be used for their ability to interrupt radical reactions.Likewise, S-and N-containing heterocycles can be useful to decompose hydroperoxides and chelate metals [22].Thus, hydrazones [23,24] and thiocarbohydrazones [25,26] with phenol fragments exhibit strong antioxidant activity.Of note is that antioxidants containing heterocyclic fragments in their chemical structure are also of considerable interest.1,2,4-Triazole-5-thione I (Figure 1) showed a higher inhibition effect of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals compared to standard antioxidant butylated hydroxytoluene (BHT), and demonstrated the in vitro inhibition of lipid peroxidation, decreasing the Fe 2+ ion-induced lipid peroxidation of essential oils [27].Pyrazole II showed activity in inhibiting the oxidation of DNA induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) [28].Compound III was found to have high radical scavenging activity, reduced the ROS levels induced by H2O2, and was also found to exhibit strong activity against lung carcinoma and human promyelocytic leukemia [29].Very few systematic studies have been conducted on structure-antioxidant activity correlations in thiazoles with phenolic fragments [30,31], but the available data show the promising antioxidant activity of this class of compound.The key intermediate for the preparation of thiazoles and many other classes of heterocycle are thiosemicarbazones, which also exhibit antioxidant properties.The relationship between the ability of thiosemicarbazone to inhibit oxidation and the coplanarity of several conjugated fragments of the molecule is known; in addition, in the case of thiosemicarbazones with a phenolic substituent, the ability to intercept radicals is influenced by the possibility of forming a hydrogen bond between the phenolic OH and the azomethine group.To investigate the influence of these factors on the antioxidant properties of the substances, a conformational analysis can be performed [32][33][34].
The aim of this study was to synthesize new antioxidants containing both phenols and a thiosemicarbazone group or thiazole and a thiazolidinone ring.The synthesized compounds were assayed for antioxidant activity to establish which substances are able to inhibit free radical processes and reduce Fe +2 ions.

Measurement and Reagents
Starting reagents and solvents were purchased from Acros and Sigma-Aldrich.3,5-Di-tert-butyl-4-hydroxybenzaldehyde [35], formylresorcinol, and 4,6-di-tert-butyl-2,3-dihydroxybenzaldehyde [36] were synthesized as described previously.A Stuart SMP30 instrument was used to determine melting points of substances.Agilent Carry 600 spectrometer was used to record the IR spectra.Spectra were obtained with attenuated total reflectance (ATR) device (ZnSe) with 32 scans.Bruker Avance II 300 spectrometer ( 1 H, 300 MHz; 13 C, 75 MHz) was used to record the 1 H and 13 C NMR spectra in DMSO D-6 with Me4Si as the internal reference.Spectra are presented in Supplementary Materials.Elemental analysis was carried out with a Vario MicroCube apparatus.

General Procedure for the Preparation of Thiosemicarbazones 1a-d
The solution of thiosemicarbazide (11 mmol) in 40 mL mixture of ethanol and acetic acid (1:1) was stirred under reflux for 3-4 h.Reaction mixture was cooled to room temperature and poured into ice water, and the precipitate formed was filtered off and recrystallized from isopropanol.

General Procedure for the Preparation of 2-Arylidenehydrazineyl-4-phenylthiazoles 2a-d
A solution of thiosemicarbazone 1 (3 mmol) with bromoacetophenone (3 mmol, 0.60 g) or bromoacetylcoumarin (3 mmol, 0.80 g) was stirred under reflux for 1.5 h in 15 mL of THF.Reaction mixture was cooled to room temperature and poured into ice water, and the precipitate formed was filtered off and recrystallized from the appropriate solvent.

Computational Details
Gaussian programs package was used for quantum-chemical calculations [39] in combination with GaussView 6.0.16 [40], which was applied for the visualization of the obtained results.For the conformational analysis, the B3LYP functional with the 6-311+G (d,p) basis set was used, as it was previously shown [41] that it allows the most accurate results to be obtained.Vibrational analysis was performed with B3LYP DFT method in 6-311+G (2d,p) basis set for the free molecules in vacuum.Obtained theoretical values were scaled with 0.9613 factor in order to improve the agreement with the experimental data [42].

Antioxidant Activity
Antioxidant activity was studied using a SOLAR UV-Vis spectrophotometer PB 2201.

ABTS Assay
To produce radical cation (ABTS +. ), 7 mM ABTS (2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium water solution was mixed with 2.45 mM potassium persulfate.Resulting solution was allowed to stand in the dark at room temperature for 12-16 h.A dark blue color appeared.To prepare the working solution, previous solution was diluted in ethanol until its absorbance was 0.70 ± 0.02 at 734 nm.To 2.7 mL of working solutions, 300 μL of 1 mmol solution of prepared compounds in DMSO (250 μmol) was added.After 10 min, the absorbance at 734 nm was measured using a spectrofotometer.ABTS radical scavenging activity was calculated as follows: ABTS activity = (Dcontrol − Dsample)/Dcontrol where Dcontrol is the absorbance of ABTS radical in ethanol; Dsample is the absorbance of ABTS radical solution with sample/standard.

Ferric Ion-Reducing Capacity Assay
To prepare a solution of potassium ferricyanide 1%, 1.0 g of K3[Fe(CN)6] was dissolved in 1 mL of 1 M HCl and a small amount of water, and the volume was adjusted to 100 mL with water.To prepare a solution of ferric chloride (0.2%), 0.2 g of FeCl3•6H2O was dissolved in 1 mL of 1 M HCl and a small amount of water, and the volume was adjusted to 100 mL with water.Sodium dodecyl sulfate (SDS) solution (1%) was prepared by dissolving 1.0 g of SDS in 100 mL of water.To 100 μL of an antioxidant solution (500 μmol) was added 400 μL of EtOH (96%), 2.5 mL of H2O, 750 μL of 1 M HCl, 750 μL of ferricyanide solution (1%), 250 μL of SDS (1%) and 250 μL of FeCl3•6H2O (0.2%).The mixture was kept at 50 °C in a water bath for 20 min, allowed to cool to room temperature, and the absorbance was measured at 750 nm relative to the reagent without the test substance.

Results and Discussion
The target compounds 1-5 were prepared as it is shown on Schemes 1 and 2. Initially, the number of thiosemicarbazones was synthesized by the condensation of thiosemicarbazide and series of aldehydes: formylresorcinol-1a; vanillin-1b; 2,3-dihydroxy-4,6-ditert-butylbenzaldehyde-1c; and 4-hydroxy-3,5-di-tert-butylbenzaldehyde-1d.The IR spectra of compounds 1a-d contain absorption at 1587-1625 cm −1 , corresponding to the C=N stretching frequency, and signals near 3450-3200 cm −1 , which correspond to N-H symmetric and asymmetric stretching vibrations, which correlates with calculated and literature data [43].In the 1 H NMR spectrum of compounds 1a-d, the protons of the formyl group of the starting compounds were not presented, and in the region 7.94-8.91ppm protons of CH=N moiety were observed.To ascertain the prevalence of one particular conformer, a potential energy surface (PES) scan was applied using the B3LYP/6-311+G (d, p) approximation.The configurations of synthetic compounds, corresponding to energy minimum, were explored through the consideration of the torsion angle H5-C5-C6-C7 and torsion angle H3-N3-N4-C5.The torsion angles were rotated between 0° and 360°, and the step was 10°.The structures corresponding to the energy minimum isomers are shown in Figure 2, and the results of the PES scan are shown in Figure 3.It was observed that for φ1, the E configurations reduce the stereochemical repulsions in relation to the Z, and E-isomers are more stable for 1a and 1b.For 1d, a strong spatial interaction of thiosemicarbazide fragment is observed with both OH and tert-butyl groups, forming the most stable rotamer, with a value of φ1 = 106.5.For φ2, Z-isomers observed.Additionally, there are absorption bands of C=O vibrations in coumarin fragments between 1710 and 1716 cm −1 in the IR spectra of these compounds.
The best yields of thiazoles 3a-d were achieved by refluxing the starting thiosemicarbazones and bromodimedone in acetic acid in the presence of sodium acetate.The IR spectra of the obtained compounds show strong absorption bands of C=O stretching vibrations near 1630 cm −1 and signals of C-O stretching vibrations near 1050 cm −1 .In the NMR spectra of the obtained substances 3a-d, there are singlets near 12 ppm corresponding to the NH proton, a singlet at 2.6 and 2.2 ppm indicating the presence of two CH2group protons, and signals near 1-1.05 ppm corresponding to six protons of two methyl groups.
In order to expand the range of heterocycles with phenol fragments obtained from thiosemicarbazones, compounds 1a-d were reacted with chloroacetic acid and maleic anhydride, resulting in the corresponding thiazolidinones 4a-d and 5a-d [44,45].In the IR spectra of compounds 4a-d, there is a strong absorption of C=O stretching vibrations at between 1690 and 1720 cm −1 .In the 1 H NMR spectra of these compounds, no peaks of the NH2 group were observed; instead, there was a singlet of the CH2 group near 3.8 ppm.In the IR spectra of thiazolidinones 5, two absorption bands corresponding to C=O vibrations are observed, one for the carboxylic group (1690-1710 cm −1 ) and another one for the thiazolidinone ring (1720-1730 cm −1 ); assignments were made in accordance with PED.The 1 H NMR spectra show peaks that are characteristic of the protons of the carboxyl group in the region of 11-13 ppm, as well as a triplet corresponding to the proton of the CH group in the ring and a multiplet in the region of 2.4 ppm, which appears due to the chirality of the carbon atom in the thiazolidinone ring.

Antioxidant Activity
For synthesized compounds, antioxidant activity assessment was performed via the ABTS radical removal method.In the assay, ABTS radical cations are generated from the ammonium ABTS salt using a strong oxidizing agent.The radical cation has an intense absorption band at 734 nm; when an antioxidant is added, the concentration of radical cations decreases, which is determined photometrically [46][47][48].The results are given in Figure 5 in terms of inhibition percentages calculated against concentration using BHT as the standard.Most of the compounds showed better results compared to the standard.The exceptions were samples 2c, 3b, 3c, 4a, 4d, and 5d.Compounds 1c, 4c, and 5a-c exhibited the best results.
In the series of obtained thiosemicarbazones 1a-d, the best results were shown by the 2,6-di-tert-butylphenol derivative 1c, which can be explained by the formation of a stable di-tert-butylphenoxyl radical.Vanillin's thiosemicarbazone 1b showed the poorest result.The antioxidant activity of diatomic phenol derivatives 1a and 1d turned out to be intermediate.This can be explained by the interaction of the phenolic hydroxyl group and the CH=N fragment at the ortho-position.Formylresorcinol's thiosemicarbazone 1a showed slightly higher activity compared to the butylated pyrocatechol derivative 1d due to the coplanarity of the 1a molecule.
Among thiazoles with phenyl and coumaryl substitutes, the same pattern was observed as for thiosemicarbazones: compound 2b, based on 2,6-di-tert-butylpnenol exhibited the highest activity, and the effectiveness of thiazole 2a based on phenylresorcinol was higher than for butylated pyrocatechol derivative.In general, the activity of derivatives of this series was lower than that of the corresponding thiosemicarbazones.In the case of thiazoles with a coumarin fragment, the resorcinol derivative 2d also turned out to be more active than the pyrocatechol derivative 2e.The introduction of the coumarin fragment increased the antioxidant activity compared to thiazoles containing a phenyl substituent.
For thiazoles prepared from bromodimedone, completely different results were observed: compounds based on diatomic phenols 3a and 3d showed the highest efficiency, but just as in previous cases, the resorcinol derivative 3a turned out to be more active.
In case of thiazolidinones 4 and 5, compounds with 2,6-di-tert-buthylphenol showed the best results, while vanillin derivatives 4b and 5b exhibited intermediate activity, formylresorcinol derivatives 4a and 5a demonstrated comparable activity, and the pyrocatechol derivative 4d and 5d again showed the lowest results.In general, compounds with thiazolidinone moieties and carboxyl group 5 have higher antioxidant activity compared to other derivatives.
To assess the electron-donating capacity of prepared compounds, PFRAP assay was used.In this assay, ferricyanide reduces to ferrocyanide, and a green-blue color of the solution appears because of the Perl's Prussian blue formation [49,50].The resulted compound has an absorption peak at λ = 700 nm.The results are presented in Figure 6.The results of the PFRAP assay displayed a stronger electron-donating capacity for thiosemicarbazone 1b, thiazoles 2d, 2e, and 3c, and thiazolidinones 4d and 5d compared to BHT.An intermediate activity was found for thiazoles 2a and thiazolidinones 4b, 4c, 5a, and 5b.The rest of the synthesized compounds displayed moderate or low antioxidant capacity compared to that of BHT.Among thiazoles 2a-c with a phenyl fragment, the best antioxidant activity was observed for 2a, which contained a resorcinol fragment.At the same time, compounds 2d and 2e, with coumaryl fragments, have exhibit higher activity than other thiazoles.Among all the compounds, 3d obtained from bromodimedone and compound 3c with a 2,6-di-tert-butylphenol fragment showed the best activity, and the pyrocatechol derivative exhibited the lowest activity.Thiazolidinones 4 and 5 exhibited better antioxidant activity than thiazoles, and among compounds of this class 2,6-di-tertbuthyl phenol and butylated catechol derivatives were more active compounds in terms of their electron-donating capacity.
The resulting compounds are capable of inhibiting radical processes and exhibit ironreducing activity, which makes them promising antioxidants, including in the development of drugs that reduce oxidative stress.

Conclusions
In conclusion, a series of thiazoles and thiazolines were synthesized from four thiosemicarbazones with phenol scaffold.In terms of the ability to react with the ABTS radical cation, in the case of thiosemicarbazones, thiazoles with phenyl and coumarin substituents and thiazolidinones, derivatives of 2,6-di-tert-butylphenol showed the best activity; derivatives of formylresorcinol and vanillin occupied an intermediate position; and the lowest results were shown by derivatives of alkylated pyrocatechol.Resorcinol derivatives showed a high iron-reducing activity among thiazoles, and alkylated catechol derivatives showed a high iron-reducing activity among thiazolidinones.Thiazolidinones 5, containing a carboxyl group, showed the greatest activity in both tests.In addition, both types of activity increase with the introduction of the coumarin moiety.

Figure 1 .
Figure 1.Examples of substances exhibiting different types of antioxidant activity.

Figure 6 .
Figure 6.Ferric ion-reducing capacity of the compounds prepared.