Screening of antioxidant and anti-infl ammatory activities among thiopyrano[2,3-d]thiazoles

The aim of present research was the investigation of antioxidant and antiexudative activities of the series of thiopyrano[2,3-d]thiazoles synthesized based on cinnamic acid amides. Methods. Organic synthesis; spectral methods; free radical scavenging assay (DPPH test); evaluation of antiexudative activity (carrageenan oedema model in rats). Results. The evaluation of the free radicals scavenging activity and antiexudative activity of series of the 2-oxo-5-phenyl-7-aryl(hetaryl)-3,7-dihydro-2H-thiopyrano[2,3-d] thiazole-6-carboxylic acid amides was performed. Among the tested compounds, rel-(5R,6S,7S)-N-(4methylphenyl)-7-(4-methylphenyl)-2-oxo-5-phenyl-3,5,6,7-tetrahydro-2H-thiopyrano[2,3-d]thiazole-6carboxamide possessed the highest level of both activities. The experimental data most probably indicate a pronounced effect of methyl groups (phenyl fragments) on the realization of the anti-infl ammatory and antioxidant effects and allow a suggestion about the antiradical mechanism of anti-infl ammatory activity of the target compounds. Conclusions. The anti-infl ammatory and free radicals scavenging activities of some tiopyrano[2,3-d]thiazoles have been established and the most active compounds were identifi ed. Some fi ndings of the structure-activity relationship were set up.


Introduction
The oxidant and free radical processes are in focus of the research.The reactive oxygen and nitrogen species such as hydroxyl and peroxyl radicals, the superoxide anion etc., are constantly produced as a result of metabolic reactions in living systems and are involved in numerous processes, both normal and pathological [1].Living systems are protected from the oxidative damage by these reactive species by enzymes (for instance, superoxide dismutase, glutathione peroxidase) and by antioxidant compounds such as ascorbic acid, tocopherols, and carotenoids, etc. [1,2].However, when the free-radical production exceeds the antioxidant capacity, these radical species attack macromolecules, thus damaging structural integrity and function of cell membranes, enzymes, and genetic material.A growing body of evidence indicates that various pathological conditions, including cardiovascular disease, arthritis, infl ammation, cancer, and Alzheimer's disease are associated, at least in part, with the damaging effects of uncontrolled free-radicals production [3,4].Thus, many biologically active compounds, especially natural, may provide the protection against mentioned processes through multiple effects, which are still poorly understood [5][6][7].These compounds may act as antioxidants by reacting with free radicals and thus interrupting the propagation of new free radical species, by chelating metal ions, etc.They also may protect against oxidant-mediated infl ammation and tissue damage by a virtue of their ability not only to scavenge free radicals but also to inhibit the activation of NF-kB (and possibly other oxidantsensitive transcription factors) [4,5] and improve certain immune responses [8].
On the other hand, many synthetic heterocycles possess similar biological profi les.For instance, 4-thia zolidinones and related heterocycles showed the antiinfl ammatory, antitumor, antitrypanosomal, antidiabetic activities, which often are associated with the antioxidant effect [9][10][11].Majority of 4thiazolidione-based lead-compounds and drug-candidates belong to the 5-ylidene-4-thiazolidinone subtype and have many advantages in the drug discovery [9,[12][13][14][15][16].At the same time, they are also characterized by some negative features.5-Ylidene-4-thiazolidinones can be considered as electrophilic and potentially reactive substances due to the possible Michael addition of the nucleophilic protein residues to the exocyclic double bond.This characterizes 5-arylidene-4-thiazolidinones as the frequent hitters or pan assay interference compounds that are useless in the modern drug discovery process because of low selectivity [17].
One of the possible solutions of such confusion may be the annealing of the mentioned 5-ylidene-4thiazolidinones into the fused heterocycles.These fused 4-thiazolidinone-based derivatives and in particular thiopyrano [2,3-d]thiazoles are considered as the cyclic isosteric mimetics of their synthetic precursors -5-ylidene-4-thiazolidinones without the Michael accepting functionalities [19,20].Moreover, design of the mentioned fused heterocycles employed the combination of thiazolidinones and other chemicals including those possessing signifi cant biological activity, for instance cinnamic acid and its derivatives (Fig. 1.), that can be treated as a benefi t in the hybride molecules design.
Therefore, as the continuation of our previous studies [19][20][21][22] in the fi eld of biologically active fused 4-thiazolidinone-based heterocycles the aim of the manuscript was the search for new compounds with antioxidant and antiexudative activities among thiopyrano [2,3-d]thiazoles.

Materials and Methods
All chemicals were of the analytical grade and commercially available.All reagents and solvents were used without further purifi cation and drying.
Free radical scavenging assay.The scavenging effect of the synthesized compounds on the DPPH (1,1diphenyl-2-picrylhydrazyl radical) was evaluated according to the Blois method [23].The solution of DPPH (150 μM) in ethanol (4 mL) was mixed with the tested compound or ascorbic acid as the referent compound (250 μM) solution in ethanol (0.2 mL).The reaction mixture was thoroughly mixed and incubated 30 min at rt. in the dark.A decrease in absorbance of the mixture (in comparision with the sample which contains only DPPH solution) was measured at 540 nm.All tests and analyses were undertaken in three replicates and the results avera ged.The scavenging activity was calculated: (%) = ([A DPPH -A s ] / A DPPH ) × 100 %, where: A DPPH is an absorbance of DPPH solution, A s is an absorbance of the tested sample.
Antiexudative assay.For antiexudative test the male rats weighing 180-220 g were used.The carragenan-induced hind paw oedema was produced by the method of Winter et al. [24].Carrageenan solution (1.0 % in sterile 0.9 % NaCl) was injected subcutaneosly into the subplanar region of the hind paw (0.1 mL to each paw) 1h after administration of the test compound.The synthesized compounds were intraperitoneally injected in a dose 100 mg/kg (in saline solution with one drop of Tween-80 TM ).Dic lofenac sodium (10 mg/kg) and ketorolac tromethamine (10 mg/kg) were used as reference compounds.Control rats received only saline solution with one drop of Tween-80 TM .The hind paw volume was measured with an electronic onkograph immediately before and 4h after carrageenan injection.The effect of test compounds on a decrease of paw oedema was compared with that control.The antiexudative activity was expressed as a decrease of rats paw oedema and was given in percentage.

Results and Discussion
The structure of new thiopyrano [2,3-d]thiazoles (fi g. 2) was designed based on the cinnamic acids deriva-Scheme.General synthetic scheme tives and the mentioned 5-ylidene-4-thiazolidinones.The synthetic protocol involved the regioselective and diastereoselective hetero-Diels-Alder reaction.The synthesized compounds were screened for anti-infl ammatory and antioxidant activities.The re is a thesis that an increase of reactive radical species levels causes the development of infl ammation along with the antioxidant effect of many antiinfl ammatory agents.Therefore, it is apparent that in addition to promoting cytotoxicity, reactive oxygen metabolites may also initiate and/or amplify infl ammation via the upregulation of several different genes involved in the infl ammatory response, such as those that code for proinfl ammatory cytokines and adhesion molecules [4].
Consequently, the compounds with antioxidant properties could be expected to offer protection in the infl ammation process.It is therefore evident that the treatment of the above mentioned pathophysiologies could benefi t from the use of drugs with both antioxidant and anti-infl ammatory activities.It has already been proven for a number of commercially available non-steroidal anti-infl ammatory drugs (NSAIDs), for example, tolfenamic acid which simultaneously possess radical scavenging properties   [25,26].Additionally, several thiazolidinone derivatives are also pronounced anti-infl ammatory and antioxidant agents and might be an exellent basis in searching for the drug-like molecules [27].
To investigate some aspects of the structure-activity relationship among thiopyrano [2,3-d]thiazoles, the antioxidant activity was assessed by the DPPHfree radicals cavenging assay and summarized in Table 1.
Among the tested compounds the rel- (7) was found to be the most active; for other derivatives, a slight antioxidant activity was observed.The comparison of the activity of compounds bearing different amide fragments (compounds 4, 6, 7 & 9) allowed the suggestion that the presence of methyl groups (including amide fragment) in compound 7 was crucial for the antioxidant activity realization.The same effect was observed in fl avonoids row, where the methyl derivatives were much more active in comparison with the derivatives bearing other substituents [28].This effect is also associated with improved lipophilicity parameter [29].Moreover, the presence of electron donating groups (especially methyls) at the aromatic core has previously been shown to infl uence strongly the redox potential of α,β-unsaturated aromatic compounds.The altered reduction potential of these compounds has an effect on the lipid peroxidation reactions, in which they can participate, and this, in turn, alters their ability to scavenge deleterious oxy radicals [30].The replacement of the methyl group in the aryl fragment (position 7 of the main core) (compounds 7, 8 & 11) to the methoxy group or a halogen atom leads to the loss of activity.
The in vivo anti-infl ammatory effect of the tested compounds was assessed by using the functional model of carrageenan-induced rat paw oedema (Table 2).
All tested compounds exhibited different protection against the carrageenan-induced paw edema.The compounds 3 and 7 were the most active among the tested thiopyrano [2,3-d]thiazoles.The antiexudative activity comparable to the reference drugs was observed in the compounds with two methyl groups (7) or in the presence of methyl and sulfamoyl groups (3).The presence of other group in the molecule led to a decrease in the activity level.The comparison of anti-radical and anti-infl ammatory activities allows us to assume the involvement of antioxidant mode in the development of thiopyrano [2,3-d]thiazoles antiinfl ammatory action.

Conclusion
The present study has shown that certain thio py rano [2,3-d]thiazoles possess low to moderate antiinfl ammatory and antioxidant activities, whereas the compound 7 was found to be a promising free radicals scavenger.The anti-infl ammatory assay led to the identifi cation of two active molecules (compo unds 3 & 7).These compounds constitute an interesting template for the design of new synthetic NSAIDs.Despite that the anti-infl ammatory mechanism was not elucidated, the mode of antiinfl ammatory action of the synthesized compounds seems to be related with their radical scavenging activity.

Fig. 2 .
Fig. 2. The structure of compounds involved into the study