Synthesis and biological evaluation of the new 1,3-dimethylxanthine derivatives with thiazolidine-4-one scaffold

Background The xanthine structure has proved to be an important scaffold in the process of developing a wide variety of biologically active molecules such as bronchodilator, hypoglycemiant, anticancer and anti-inflammatory agents. It is known that hyperglycemia generates reactive oxygen species which are involved in the progression of diabetes mellitus and its complications. Therefore, the development of new compounds with antioxidant activity could be an important therapeutic strategy against this metabolic syndrome. Results New thiazolidine-4-one derivatives with xanthine structure have been synthetized as potential antidiabetic drugs. The structure of the synthesized compounds was confirmed by using spectral methods (FT-IR, 1H-NMR, 13C-NMR, 19F-NMR, HRMS). Their antioxidant activity was evaluated using in vitro assays: DPPH and ABTS radical scavenging ability and phosphomolybdenum reducing antioxidant power assay. The developed compounds showed improved antioxidant effects in comparison to the parent compound, theophylline. In the case of both series, the intermediate (5a–k) and final compounds (6a–k), the aromatic substitution, especially in para position with halogens (fluoro, chloro), methyl and methoxy groups, was associated with an increase of the antioxidant effects. Conclusions For several thiazolidine-4-one derivatives the antioxidant effect of was superior to that of their corresponding hydrazone derivatives. The most active compound was 6f which registered the highest radical scavenging activity.Graphical abstract Design and synthesis of new thiazolidine-4-one derivatives. Electronic supplementary material The online version of this article (doi:10.1186/s13065-017-0241-0) contains supplementary material, which is available to authorized users.

Diabetes mellitus is a chronic metabolic disorder considered a major health problem in the whole world. Every year 4 million people die from diabetes mellitus and 1.5 million new cases are diagnosed. This disease is characterized by hyperglycemia, a condition which, if not properly controlled, can lead to complications at the level of different organs. It mainly affects the eyes, the heart, the kidneys and the blood vessels. Hyperglycemia also generates reactive oxygen species (ROS) which can produce cell damages by means of different mechanism [27]. It has been proven that oxidative stress (an imbalance between the production of ROS and the scavenging ability of the body) holds a key role in the development of diabetes mellitus and its complications. The scavenging ability is closely related to the concentration of endogenous oxidative enzymes such as catalase, glutathione peroxidase and superoxide dismutase [28].
In order to develop new compounds with antioxidant effects and potential applications in antidiabetic therapy, new thiazolidine-4-one derivatives with xanthine structure have been synthesized. The structure of these compounds was proved by means of spectral methods (FT-IR, 1 H-NMR, 13 C-NMR, 19 F-NMR, HRMS) and their antioxidant effects were evaluated using in vitro assays: DPPH and ABTS radical scavenging ability and phosphomolybdenum reducing antioxidant power assay.

Chemistry
The new 1,3-thiazolidine-4-one derivatives were synthesized according to Scheme 1. Theophylline (1,3-dimethylxanthine) 1, in the presence of sodium methoxide, gave the salt 2 in a quantitative yield; the salt in its turn reacted with ethyl chloroacetate and resulted in theophylline-ethyl acetate 3 [4]. The reaction of the compound 3 with an excess of hydrazine hydrate 64% resulted in a very good yield of theophylline hydrazide 4. Then, the condensation of the compound 4 with different aromatic aldehydes led to the formation of the corresponding hydrazones 5a-k in satisfying yields [29,30]. Finally, the cyclization of hydrazones (5a-k) in the presence of mercaptoacetic acid had as result thiazolidine-4-one derivatives 6a-k in moderate to excellent yields (Table 1). Totally were obtained 22 compounds from which 19 are new (8 hydrazones: 5b, 5d, 5e, 5g-5k and 11 thiazolidine-4-ones: 6a-k).
The structure of the compounds was proved on the basis of the spectral data (IR, 1 H-NMR, 13 C-NMR, 19 F-NMR, HRMS) provided in the "Experimental section" part of the paper. The IR and NMR spectral data for compounds 3 and 4 were previously presented [4].
In the 1 H-NMR spectra of hydrazones 5a-k there were identified two sets of signals which corresponded to the two tautomer forms and were in dynamic equilibrium with each other. The proton from amide group (CO-NH) is responsible for the lactam-lactim tautomerism, obtaining two forms: the hydrazone (lactam form, HN-C=O) and the tautomer (lactim form, N=C-OH). The ratio between tautomers ranged between 9:1 and 7:3, depending on the compound. The proton of the azomethine group (N=CH) resonated as a singlet at 7.96-8.38 ppm for one form and at 8.06-8.55 ppm for the other form. The proton of the amide group (CO-NH) appeared as a singlet at 11.55-11.83 ppm in the case of the hydrazone and at 11.63-11.83 ppm in the case of the tautomer form. The tautomerism was proved by 1 H-NMR at 80 °C, when one set of signals was recorded.
The success of the cyclization process which resulted in the formation of the thiazolidine-4-one ring, was proved by means of 1 H-NMR data. The proton from the N-CH-S group was recorded as a singlet between 5.75 and 6.10 ppm while the protons of the methylene group (CH 2 -S) resonated as doublets of doublets or multiples in the interval between 3.63 and 3.80 ppm.
The structure of the synthesized compounds was strengthened by 13 C-NMR data. The compounds 5a-k had two azomethine groups (N=CH), one from the theophylline part and another one from the hydrazone chain. The carbon signals of these groups were observed between 140.3 and 148.8 ppm. Moreover, the carbons from the thiazolidine-4-one ring appeared at 56.9-61.7 ppm (N-CH-S) and 29.9-30.1 ppm (CH 2 -CO).
The fluorine atom from the structure of 5d and 6d, resonated in 19 F-NMR spectra as a specific signal registered at −110.5 and −110.4 ppm in the case of the tautomer forms of hydrazone and at −110.2 ppm in the case of the thiazolidine-4-one derivatives.
The molecular mass of hydrazones 5a-k and of the corresponding thiazolidine-4-one derivatives, 6a-k, was probed by means of high resolution spectral mass. The spectral mass data coupled with the NMR data ( 1 H-NMR, 13 C-NMR, 19 F-NMR) proved the proposed structure for all synthesized compounds.

Biological evaluation DPPH radical scavenging assay
2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is usually applied for the evaluation of the antioxidant activity of different compounds. The method is based on the reduction of DPPH, which is violet in methanol solution, to a pale yellow compound, under the action of an antioxidant (proton donating agent). The absorbance of the yellow form is measured at 517 nm [31,32]. The DPPH radical scavenging ability (%) of the theophylline-acethydrazide derivatives 5a-k was calculated at different concentrations (0.4, 0.8, 1.2, 1.6, 2.0 mg/mL). Higher values of the scavenging ability indicate a superior effectiveness of the scavenging radical potential. It was observed that the scavenging ability of the hydrazones increased with the concentration, the best inhibition rate being recorded at the highest concentration used (2 mg/mL). The most significant increase from 0.4 to 2 mg/mL was recorded for 5a (R=H). At the highest concentration used (2.0 mg/ mL) the inhibition rate ranged from 4.51 ± 0.36% for 5c (R = 4-Cl) to 18.65 ± 0.43% for 5a (R=H) ( Table 2). The inhibition rate of 5a was higher than that of theophylline (1, 12.14 ± 0.20%). The compounds 5d (R=4-F), 11.65 ± 0.19% and 5g (R=3-OCH 3 ), 10.66 ± 0.19% showed a similar activity to theophylline. However, the hydrazone derivatives were less active than vitamin C which was used as positive control.
The scavenging ability of the theophylline-acethydrazide derivatives was improved by cyclization to the corresponding thiazolidine-4-one derivatives 6a-k. Their scavenging ability at different concentrations (0.4, 0.8, 1.2, 1.6, 2.0 mg/mL) was higher than the value recorded for the corresponding hydrazones. The inhibition rate of 6a-k was similar to the one of 5a-k and increased with the concentration, the best inhibition rate being recorded at the highest concentration used (2 mg/mL). At this concentration the best inhibition rate was shown by 6c (R=4-Cl) and 6k (R=4-CH 3 ), with vlues of 77.53 ± 0.47% (EC 50 = 1.1640 ± 0.0123 mg/mL) and 68.28 ± 0.19% (EC 50 = 1.4389 ± 0.0130 mg/mL), respectively. In comparison to theophylline (1) these compounds were about 6.5 times and six times more active. The most promising compound seemed to be 6f (R=4-OCH 3 ), which had an inhibition rate of 64.50 ± 0.59% at 0.3 mg/mL, showing the best EC 50 value (0.2212 ± 0.0011 mg/mL) ( Table 3). These data supported the conclusion that the presence of the methoxy, chloro and methyl group in the para position of the phenyl ring of the thiazolidine-4-one scaffold had a good influence on the radical scavenging activity.
A good influence was also showed by the presence of the fluoro group in para position and of the methoxy group in ortho and meta position; the corresponding compounds 6d (R=4-F), 6e (R=2-OCH 3 ), 6g (R=3-OCH 3 ) and 6j (2,3-OCH 3 ) being about three time more active than theophylline. However all tested compounds were less active than Vitamin C used as positive control.

Phosphomolybdenum reducing antioxidant power (PRAP) assay
Phosphomolybdenum reducing antioxidant assay, known as total antioxidant capacity assay, is a spectrophotometric method based on the formation of green colored phosphomolybdenum complex after the reduction of Mo(VI) to Mo(V) under the action of electron donating compounds in acidic medium [34]. An increase in optical density means a better total antioxidant capacity. In Figs. 1 and 2 there were presented the absorbance values of the tested compounds (5a-k, 6a-k) at different concentrations (0.0291, 0.0582, 0.0872, 0.1163 and 0.1745 mg/mL). As we expected, the absorbance of the tested compounds increased with the concentration, the highest value of absorbance/activity being recorded at 0.1745 mg/mL, the highest concentration used. The data expressed as EC 50 values (mg/mL) were shown in Tables 8 and 9.

Experimental section General procedures
The melting points were measured by using a Buchi Melting Point B-540 apparatus and they were uncorrected. The FT-IR spectra were recorded on a Thermo-Nicolet AVATAR 320 AEK0200713 FT-IR Spectrometer, at a resolution of 4 cm −1 after six scans in the 4000-500 cm −1 . The spectra processing was carried out with the Omnic Spectra Software. The 1 H-NMR (250, 400 MHz), 13 C-NMR (63, 101 MHz) and 19 F-NMR (376 MHz) spectra were obtained on two types of Bruker Avance spectrometer: 250 and 400 MHz, using tetramethylsilane as internal standard and DMSO-d 6 and CDCl 3 as solvents. The chemical shifts were shown in δ values (ppm). The mass spectra were registered by using a BrukerMaXis Ultra-High Resolution Quadrupole Time-of-Flight Mass Spectrometer. The reactions were monitored by TLC, using pre-coated Kieselgel 60 F254 plates (Merck, Whitehouse    Station, NJ, USA) and the compounds were visualized using UV light. The absorbance for biological assays was measured using a GBC Cintra 2010 UV-VIS spectrophotometer at different wavelengths: 517, 734 and 695 nm. The values were recorded in Cintral Software.

Synthetic procedures
The synthesis of hydrazide derivatives (5a-k) The general procedure used for the synthesis of theophyllineacethydrazide derivatives and a part of their physical and chemical characteristics were described in our previous papers [29,30]. The synthesis of the theophylline sodium salt 2, theophylline ethyl acetate 3 and theophylline acetyl-hydrazine 4, used as intermediaries in the synthesis of hydrazone derivatives 5a-k was performed according to the literature procedure and some of their physical and chemical characteristics were presented in our previous paper [4]. N-Benzylidene-2-(1,3-dimethylxanthin-7-yl)acethydrazide (5a) Tautomeric mixture (8:2). 13

Synthesis of the theophyllinyl-acetamido-thiazolidin-4-one derivatives (6a-k)
Hydrazide derivatives (5a-k) (5 mmol) were reacted with thioglycolic acid (100 mmol) using freshly distillated toluene as solvent, according to the procedure described for other thiazolidine-4-one derivatives [35]. The mixture was heated under reflux and stirred at 120 °C for 18 h. The reaction was monitored by Thin Layer Chromatography (TLC), in UV light at 254 nm, using ethyl acetate: methanol (9.6:0.4, v/v) as eluent system. At the end of the reaction, the solvent was removed and the mixture was cooled at 0 °C on ice bath. After that, dichloromethane (100 mL) was added and the mixture was neutralized, under continuous stir-ring at 0 °C, with sodium bicarbonate 10%. The organic layer was separated and washed with alkaline solution (two times with 100 mL) and then acidulated with hydrochloric acid 10% (300 mL). Finally, the organic phase was dried on anhydrous MgSO 4 , and it was concentrated by rotary evaporator under reduce pressure. The residue was purified on silica gel column, using ethyl acetate as eluent solvent.

Biological evaluation
The antioxidant activity was estimated using in vitro tests: DPPH and ABTS radical scavenging ability and total antioxidant capacity.
DPPH radical scavenging assay The antiradical activity against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was measured as described in [32] with slight modifications. Different samples of the tested compounds (50, 100, 150, 200, 250 μL) from a stock solution (20 mg/mL in DMSO) were mixed with DMSO to obtain 1300 μL, and then DPPH in methanol (1200 μL, 15 μM) was added. The final concentrations of the compound in the test tube were 0.4, 0.8, 1.2, 1.6, and 2 mg/mL, respectively. The mixture was left for 30 min in the darkness and, after that, the absorbance was measured at 517 nm against a blank solution (methanol). The radical scavenging capacity was calculated using the following equation: where A control is the absorbance of the mixture of 1300 μL DMSO and 1200 μL DPPH after 30 min; A sample is the absorbance of the sample after 30 min.
For the most active compounds, the effective concentration (EC 50 ) was calculated by linear regression analysis. The theophylline (parent compound) and ascorbic acid (vitamin C) were used as reference and positive control, respectively. All tests were performed in triplicate.
ABTS radical scavenging assay ABTS + radicals were activated by reacting of ABTS (2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid)) (7 mM) with ammonium persulphate (2.45 mM) and the mixture was left for 16 h in the darkness, at room temperature. The resulted ABTS ·+ solution was diluted with ethanol to obtain an absorbance value of 0.7 ± 0.02 at 734 nm as described in [33]. Different samples of the tested compounds (50, 100, 150, 200 μL) from a stock solution (10 mg/mL in DMSO) were mixed with DMSO to obtain 200 μL, and then freshly ABTS ·+ solution (1800 μL) was added. The final concentrations of the compound in the test tube were 0.25, 0.50, 0.75 and 1 mg/mL, respectively. The mixture was stirred and left in the dark for 6 min., at room temperature, and the absorbance was measured at 734 nm. The radical scavenging capacity was calculated according to the following equation: where A control is the absorbance of the mixture of 200 μL DMSO and 1800 μL ABTS ·+ after 6 min; A sample is the absorbance of sample after 6 min.
For the most active compounds the effective concentration (EC 50 ) was calculated by linear regression analysis. The theophylline (parent compound) and ascorbic acid (vitamin C) were used as reference and positive control, respectively. All tests were performed in triplicate.
Phosphomolybdenum reducing antioxidant power (PRAP) assay The phosphomolybdenum method was used according to the procedure described in [34] with few Inhibition percent % = A control − A sample /A control × 100.
Scavenging activity % = A control − A sample /A control × 100.
modifications. Different samples of the tested compounds (25,50,75, 100, 150 μL) from a stock solution (2.5 mg/ mL in DMSO) were mixed with DMSO to obtain 150 μL, and then 2 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, 4 mM ammonium molybdate) were added. The test tubes were closed and incubated in the oven at 95 °C, for 90 min. After cooling at room temperature, the absorbance was measured at 695 nm against a blank solution (DMSO with 2 mL reagent solution). The final concentrations of the compound in the test tube were 0.0291, 0.0582, 0.0872, 0.1163 and 0.1745 mg/mL. EC 50 was calculated for all the tested compounds. Theophylline (parent compound) and ascorbic acid (vitamin C) 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 by mean ± SD. The EC 50 values were calculated by linear interpolation between the values registered above and below 50% activity.

Conclusions
In this research new xanthine derivatives based on thiazolidine-4-one scaffold have been synthetized. The intermediate and final compounds were characterized in terms of physical properties and their structure was proved using spectral methods (FT-IR, 1 H-NMR, 13 C-NMR, 19 F-NMR, HRMS). The antioxidant potential was evaluated using in vitro assays: DPPH and ABTS radical scavenging ability and phosphomolybdenum reducing antioxidant power assays. Some of the thiazolidin-4-one derivatives showed improved antioxidant effects in comparison to the corresponding hydrazones and parent compound, theophylline. A good antiradical scavenging effect (DPPH, ABTS) was shown by 6f (R=4-OCH 3 ), 6d (4-F), 6c (4-Cl) and 6k (4-CH 3 ). The compounds 6c and 6k showed also a high phosphomolybdenum reducing antioxidant power. The preliminary results support the antioxidant potential of some thiazolidine-4-one derivatives and motivate our next research focused on streptozotocin-induced diabetic rat model.