Synthesis, Molecular Docking and Biological Properties of Novel Thiazolo[4,5-b]pyridine Derivatives

The synthesis, anti-inflammatory and antioxidant properties of novel 5-hydroxy-7-methyl-3H-thiazolo[4,5-b]pyridin2-one derivatives were discussed. Fused thiazolo[4,5-b]pyridin-2-ones were synthesized and modified at the N3, C5 and C6 positions of the main core in order to obtain the compounds with a satisfactory pharmacological profile. The synthesized compounds were preselected via molecular docking for further testing of their anti-inflammatory activity in vitro. Evaluation of novel compounds over the carageenin induced rat paw edema revealed strong anti-inflammatory action of some compounds including (thiazolo[4,5-b]pyridin-3(2H)-yl) propanenitrile (5) and thiazolo[4,5-b]pyridin-3(2H)-yl) propanoic acid (6) even exceeding the standard – Ibuprofen. The antioxidant activity of the synthesized compounds was measured in vitro by the method of scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.


Introduction
Inflammation is an essential response of living organisms to the common ailments starting from traumatic disorder or fever associated with infection to major life-threatening diseases like myocardial infarction or brain haemorrhage or infarct. 1 Literature data survey has revealed that numerous nonsteroidal anti-inflammatory drugs (NSAIDs), which belong to different chemical classes, have been developed to treat inflammatory disorders. During the past few years, the long-term use of NSAIDs has been severely hampered by the emerging of several serious effects such as gastrointestinal ulcers, hepatotoxicity, renal dysfunction, and cardiotoxicity. 2 Unfortunately all of the proposed medications provoke serious side effects. 3 In general, NSAIDs exert their pharmacological action by inhibiting the synthesis of prostaglandins (PGs) by non-selectively cyclooxygenases 1 and 2 (COX-1 and COX-2), either selective COX-2 blocking. Inhibition of COX-1 is also responsible, in part, for gastrointestinal side effects, which are the most frequent side effects of NSAIDs. 4 These conditions generate one of the biggest challenges of modern medicinal chemistry for the development of alternative anti-inflammatory drugs with minimal adverse effects. [5][6][7] No less challenging is the search for new antioxidants. Different environmental stress factors like pollution, drought, temperature, excessive light intensities, and nutritional limitation can increase the production of reactive oxygen species (ROS). [8][9] Oxidative stress is a major contributing factor for developing degenerative diseases like atherosclerosis, ischemic heart disease, ageing, diabetes mellitus, cancer and others. 10 Antioxidants can interfere with the oxidation process by reacting with free radicals, and also by acting as reactive species scavenger. 11 Therefore, various natural well and synthetic antioxidants are used to scavenge free radicals. In this regard, it is important to synthesize new classes of compounds with antioxidant properties.
The present work is devoted to the synthesis of a series of novel 3H-thiazolo [4,5-b]pyridine-2-ones by the structural modification of the core heterocycle in its N 3 , C 5 and C 6 positions for further pharmacological in vivo anti-inflammatory activity assay based on the results obtained via computer simulation -molecular docking and in vitro antioxidant screenings.

1. Materials
All chemicals were of analytical grade and commercially available. All reagents and solvents were used without further purification and drying.

Chemistry
All melting points were determined in an open capillary and are uncorrected. 1 H and 13 C NMR spectra were recorded on a Varian Mercury 400 (400 MHz for 1 H) instrument with TMS or deuterated solvent as an internal reference. Mass spectra were run using Agilent 1100 series LC/MSD, Agilent Technologies Inc. with an API-ES/APCI ionization mode. Satisfactory elemental analyses were obtained for new compounds (C ± 0.17, H ± 0.21, N ± 0.19).

3. Molecular Docking
Molecular docking was conducted with OpenEye Scientific Software program package as a computer method approach to the search of molecules with affinity to certain biotargets. Used software includes Fred Receptor, Vida, Flipper, Babel 3, Omega 2 and Fred 2 programs.

4. Anti-inflammatory Activity Evaluation Assays
Anti-inflammatory activity 39 was evaluated using carrageenan induced rat paw edema method in rats. Outbred (male/female) white rats weighing 180-220g were used for the edema test. The experiments were carried out in accordance with the requirements of the European convention for the protection of vertebrate animals used for experimental and other scientific purposes. The experimental protocol was approved by the Danylo Halytsky Lviv National Medical University ethics committee, constituted by the Ministry of Health of Ukraine.
Animals were divided into 15 groups comprising five rats per group. One group was kept as the control and remaining 14 groups (test groups) were used to determine the anti-inflammatory activity elicited by the 13 drug candidates, respectively. Rats were kept in the animal house under standard conditions of light and temperature on the general diet prior to the experiment. The standard drug, Ibuprofen (50 mg/kg body weight) and the test compounds (50 mg/kg body weight) were dissolved in DMSO and administered through intraperitoneal route. DMSO was injected to the control group. At 30 minutes later, 0.1 ml of 2 % carrageenan solution in saline was injected in the sub-plantar region of the right hind paw of each rat. After 4 h of the carrageenan injection, the volume of paw edema (in ml) was measured using water plethysmometer and decrease in paw edema was compared between the control group and the test groups. The inflammatory reaction inhibition was expressed as a percent of paw volume reduction and it was calculated using the following formula: (1) where V control is the increase in paw volume in control group animals, and V is the increase in paw volume in animals injected with the test substances.

5. Antioxidant Activity Evaluation Assays
The antioxidant activity was determined on the basis of free radical scavenging activity of stable 2,2-diphe-nyl-1-picrylhydrazyl (DPPH). The effect of the studied compounds on DPPH radicals was estimated according to the method of Blois [40][41] with minor modifications. The solution of DPPH in ethanol with the concentration of 150 µmoles/L (4 mL) was mixed with the compound or control solution in ethanol its concentration been 250 µmoles/L (0.2 mL). The reaction mixture was vortex mixed thoroughly and incubated at room temperature in the dark for 60 min. Simultaneously, a control was prepared as ascorbic acid solution in ethanol (0.2 mL) mixed with of DPPH solution in ethanol (4 mL) without sample fraction. Reduction in the absorbance of the mixture was measured at 517 nm using ethanol as blank. Ascorbic acid was used as a standard. Also, the absorbance of DPPH solution was measured. Percentage of free-radical-scavenging activity was expressed as percent inhibition and it was calculated using the following formula: (2) where A DPPH is the absorbance of DPPH free radicals solution, and A c is the absorbance of a sample. Each experiment was performed in triplicate and average values were recorded. Results are expressed as the means ± S.D.
Furthermore, compound 1, due to the presence of hydroxyl-moiety in position 5 of thiazolo [4,5-b]pyridine core, represents a convenient reagent for thiazolo [4,5-b] pyridin-5-yl 4-carboxylates (9-14) generation via acylation reaction by chloroacetyl chloride or appropriate aromatic acyl chlorides (Scheme). Powders of these products are well soluble in DMF, DMSO and acetic acid, and sparingly soluble in water and in other organic solvents.
Structures of the obtained compounds were confirmed by 1 H and 13 C NMR spectroscopy, mass spectroscopy and elemental analysis. All these new compounds possess spectroscopic data in accordance with the proposed structures.

2. Molecular Docking
Previously we have shown a good correlation between results obtained in computer simulation using OpenEye Software with that obtained in the corresponding in vitro assays. 42,43 Crystallographic models of COX-1 and COX-2 (4O1Z and 5IKR correspondingly) were obtained from Protein Data Bank (www.rcsb.org). As research objects thiazolo [4,5-b]pyridine derivatives, common NSAIDs (aspirin, mefenamic acid, diclofenac, ibuprofen, indomethacin, ketoprofen, ketorolac, others) and well-known selective COX-2 inhibitors, such as parecoxib, lumiracoxib, etoricoxib and others, were chosen. To estimate in silico COX-2-compound and COX-1-compound binding seven scoring function values (Chemgauss 2, Chemscore, PLP, Screenscore, Shapegauss, Zapbind and Consensus) were calculated. Cumulative (Consensus) scoring function ranking allowed us to select compounds, which could prospectively be selective COX-2 inhibitors. Fred receptor program allows to extract the active sites (biotarget) of COX-2 and COX-1 from crystallographic models for molecular docking.
Molecular docking studies included generation of R-, S-and cys-trans isomers of ligands using program Flipper with further 3D optimization of isomers using program Hyper Chem 7.5 (www.hyper.com) (molecular mechanics method MM+ and semi-empirical quantum-mechanical method PM3). Conformers were generated via Omega 2. Further program Fred 2 choose minimum energy conformation for each molecule and 3D molecular docking was performed.

a) b)
Values of the seven scoring functions (Chemgauss 2, Chemscore, PLP, Screenscore, Shapegauss, Zapbind and Consensus) were obtained as a result. Ranking property (compound ranking) of the consensus scoring function, which includes values of all scoring functions, allowed to analyze the results easily.
Ranking and analysis of the molecular docking results were obtained using the selected compounds and crystallographic model of COX-2 with cumulative scoring function (consensus). Consensus results allowed us to select compounds, which could prospectively be selective COX-2 inhibitors at the level of mefenamic acid and Ibuprofen for future (in-depth) pharmacological studies for further evaluation of in vitro anti-inflammatory activity. The interactions between COX-2 active site and the most active compound 5 in comparison with selective inhibitor mefenamic acid is shown in Figure. Moreover, it should be noted that results predicted via docking correlate quite well with that obtained in the in vitro assay. The selected "lead" compound 5 based on the in vitro screening results was also predicted to be the most active in the docking studies.
On the contrast, generated conformations of thiazolo [4,5-b]pyridine derivatives did not possess the necessary parameters for successful binding to the target COX-1 active site and were found to be bad substrates of cyclooxygenase-1 during docking experiment.

Anti-inflammatory Activity in Vivo Evaluation
Carrageenan-induced paw edema is the most widely used animal model of acute inflammation. In vivo studies of novel thiazolo [4,5-b]pyridine-2-one derivatives were carried out for anti-inflammatory activity employing the carrageenan-induced rat paw edema method. The NSAID drug Ibuprofen in its effective therapeutic dose was tested in parallel as an activity reference. Results of paw edema decreasing were expressed as the mean ± standard deviation and compared statistically with the control group using Student's t-test. A level of p<0.05 was adopted as the test of significance ( Table 1). The percentage protection against inflammation was calculated as % inhibition by comparison between DMSO injected control group and drugs-tested groups.
Evaluation of anti-inflammatory activity indicated that 8 compounds (2, 3, 7, 8, 10, 11, 12 and 13) showed no significant decrease in edema; the inhibition rate for them was observed at the level of 22.6−30.1 % as compared to control group. The compounds 4, 9 and 14 possessed the anti-inflammatory activity in the range of 35.6−42.1 % which is comparable to the effect of Ibuprofen. The anti-inflammatory evaluation test for compounds 5 and 6 gave the result at the level of 45.3−48.8 % inhibition indicating that the compounds 5 and 6 were more potent than Ibuprofen.

4. In Vitro Antioxidant Assay
The antioxidant activity was determined on the basis of free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The DPPH method is described as a simple, rapid and convenient method for screening of many samples for radical scavenging activity. These advantages make the DPPH method interesting for testing newly synthesized compounds to scavenge radicals and to find out antioxidant drug candidates.
DPPH radical had found many applications due to its high stability in a methanolic solution and intense purple color. In its oxidized form, the DPPH radical has an absorbance maximum at a wavelength of 540 nm. The absorbance decreases when the radical is reduced by antioxidants. Its reduction affords 2,2-diphenyl-1-picrylhydrazine (DPPH-H), or the corresponding anion(DPPH -) in basic medium. The DPPH radical acts as a scavenger for other odd-electron species which afford para-substitution products at phenyl rings. In the present paper, we demonstrate modified spectrophotometric method making use of the DPPH radical and its specific absorbance properties. The free-radical-scavenging activity of each compound was assayed using a stable DPPH and was quantified by decolorization the solution being mixed with DHHP at a wavelength of 540 nm. The absorbance of DPPH solution in ethanol (150 mmoles/l) was measured as 0.77. The ab-sorbances and free-radical-scavenging activities % inhibitions of standard (ascorbic acid) and each compound are listed in Table 2.
The antioxidant activity evaluation results showed that, in general, most of the tested compounds possess insignificant free radical scavenging effect being in the range of 1.5%-32.0%.

Conclusions
In summary, we presented an efficient synthetic approaches to a number of thiazolo [4,5-b]pyridin-2-one derivatives for their anti-inflammatory and antioxidant activity evaluation. We have shown that the proposed synthetic protocols provided the possibility to design 5-hydroxy-7-methyl-3H-thiazolo [4,5-b]pyridin-2-ones diversity with a considerable chemical novelty involving [3+3]cyclocondensation, cyanoethylation, hydrolysis, and acylation reactions. The obtained results of the performed biological activity evaluation suggested the core fused heterocycle as a promising scaffold in anti-inflammatory drug development. On the contrary, the free radical scavenging effect was found to be insignificant. Further optimization of the structure to improve biological activity is currently in progress.