Development of effective anti-inflammatory Drug canDiDates among novel thiazolopyriDines

In an effort to develop novel anti-inflammatory agents, a series of thiazolo[4,5-b]pyridines were synthesized and modified at the N3 position. The structures of the obtained compounds were confirmed by 1h NMr spectroscopy and elemental analysis. The synthesized substances were preselected via molecular docking to be tested for their anti-inflammatory activity in vitro. Evaluation of compounds using the carrageenaninduced rat paw edema method showed strong anti-inflammatory action of some compounds (1, 2, 8) which exceeded that of ibuprofen.

In an effort to develop novel anti-inflammatory agents, a series of thiazolo [4,5-b]pyridines were synthesized and modified at the N 3 position. The structures of the obtained compounds were confirmed by 1 h NMr spectroscopy and elemental analysis. The synthesized substances were preselected via molecular docking to be tested for their anti-inflammatory activity in vitro. Evaluation of compounds using the carrageenaninduced rat paw edema method showed strong anti-inflammatory action of some compounds (1,2,8) which exceeded that of ibuprofen.
introduction Inflammation is one of the common events in the majority of acute as well as chronic debilitating diseases and represents a chief cause of morbidity in the contemporary era of modern lifestyles. Current approaches to overcome inflammation include the use of nonsteroidal anti-inflammatory drugs (NSAIDs), immune selective anti-inflammatory derivatives, selective glucocorticoid receptor agonists, resolvins/protectins, and TNF inhibitors. Although drug treatment has been improved to some extent, it is still yet a challenge for pharmaceutical chemists to explore more effective and potent therapeutic agents to treat inflammation and reduce the signs and symptoms of acute inflammation and chronic inflammatory diseases [1].
The use of scientific and technological innovations as a research tool combining multidisciplinary knowledge in informatics, biotechnology, chemistry and biology are essential for optimizing time and reducing costs in drug design [2]. The integration of these in silico techniques makes it possible to search for new anti-inflammatory drugs available drugs.
The present work is devoted to the synthesis of a series of novel thiazolo [4,5-b]pyridine-2-ones for further pharmacological in vivo anti-inflammatory activity assay based on the results obtained from computer simulation of molecular docking.
were used without further purification and drying. Ibuprofen was purchased from the medical store.
Chemistry. All melting points were determined in an open capillary. The elemental analysis experimental data on contents of sulfur and nitrogen were within ±0.3% of the theoretical values. 1 H NMR spectra of synthesized compounds in dimethyl sulfoxide (DMSO)-d6 solutions were recorded on a spectrometer Varian Mercury VX-400 [Agilent Technologies, San Francisco, USA] (400 MHz) at 298 K. Chemical shifts are reported as δ (ppm) relative to tetramethylsilane (TMS) as an internal standard . The coupling constant J is expressed in Hz.

b]pyridin-3(2H)-yl)-N-aryl-l propanamides (Compounds 3-10).
A mixture of the propanoic acid (Compound 2, 10 mmol), thionyl chloride (57 mmol), and dioxane (30 ml) was placed into the round-bottomed flask. The reaction mixture was refluxed for 30 min and the product was precipitated with n-hexane, then the precipitate was filtered off. The resulting acyl chlorides are used for further transformations without further purification. The obtained 3-(5-hydroxy-7-methyl-2-oxothiazolo[4,5-b]pyridin-3(2h)-yl) propanoyl chloride (10 mmol) was dissolved in anhydrous dioxane (10 ml), an appropriate aromatic amine (10 mmol), and triethylamine (10 mmol) were added to the solution. The reaction mixture was refluxed for 15 min. Upon cooling, the mixture was diluted with water, the precipitated crystalline solid was filtered off, washed with methanol and dried. The obtained compounds were recrystallized from acetic acid.  Pharmacology. Anti-inflammatory activity [22] was evaluated using the carrageenan-induced rat paw edema method in Wistar rats (weight 180-220 g). 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. Ethical Committee or Institutional Animal Care and Use Committee Approval: 18/03/2013 No 3.

3-(5-Hydroxy-7-methyl-2-oxothiazolo[4,5-b] pyridin-3(2H)-yl)-N-phenylpropanamide (Com
Animals were divided into 12 groups comprising five rats per group. One group was kept as the control and the remaining 11 groups (test groups) were used to determine the anti-inflammatory activity elicited by ibuprofen and the 10 compounds. Rats were kept in the animal house under standard conditions of light and temperature on a standard 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 an intraperitoneal route. DMSO was injected into the control group. At 30 minutes later, 0.1 ml of a 2% carrageenan solution in saline was injected in the sub-plantar region of the right hind paw of each rat. At 4 h after the carrageenan injection, the volume of paw edema (in ml) was measured using a water plethysmometer [Orchid Scientific, Mumbai, India] and decrease in paw edema was compared between the control group and the test groups.
Results of decreased paw edema 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 considered to be significant. The inflammatory reaction inhibition was expressed as a percent reduction of paw volume and was calculated using the following formula: where V control is the increase in paw volume in control group animals; V is the increase in paw volume in animals injected with the test substances.
The high electrophilicity of the basic scaffold N 3 position makes it possible to use its functionalization as a fairly convenient method for obtaining a variety of N 3 -substituted derivatives, thereby extending the number of thiazolo [4,5-b]pyridines.
For the compound 2 carboxyl group transformation, the corresponding chloranhydride was obtained, which belongs to unstable highly reactive reagents, so its application in further transformations was carried out without isolation by introducing aromatic amine acylation. The above conversion allowed us to obtain a number of suitable propionamides (compounds 3-10) (Scheme). Powders of these products are well soluble in DMF, DMSO, and acetic acid but sparingly soluble in water and other organic solvents.
The structures of the obtained compounds were confirmed by 1 H spectroscopy and elemental analysis. All these new compounds gave spectroscopic data in accordance with the proposed structures.

Scheme. Synthesis of some thiazolo[4,5-b]pyridines
were obtained from the Protein Data Bank (www. rcsb.org). The following were chosen 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 (parecoxib, lumiracoxib, etoricoxib and others). To estimate in silico COX-1-compound and COX-2-compound binding, values of the scoring function were calculated. The Chemgauss 4 scoring function ranking allowed us to select compounds which could prospectively be selective COX-2 inhibitors. The Fred receptor program allowed us to extract the active sites (biotargets) of COX-1 and COX-2 from crystallographic models for molecular docking.
Molecular docking studies included generation of R-, S-and cys-trans isomers of ligands and then conformers were generated using the Omega 2 program with Flipper parameter. In addition, the Hybrid program that uses elements of ligand based design was used to enhance performance. Typically, protein structure is determined with X-ray crystallography in the presence of a known binding ligand (or bound ligand). The Hybrid program uses the information present in both the structure of the protein and the bound ligand to enhance docking performance. Values of the scoring function (Chemgauss 4) were obtained as a result. The ranking property of the scoring function allowed us to easily analyze the results (Table 1).
Ranking and analysis of the molecular docking results were obtained using the selected compounds and crystallographic model of COX-2 with scoring function (Chemgauss 4). The results allowed us to select compounds, which could prospectively be selective COX-2 inhibitors at the level of diclofenac and ibuprofen for future (in-depth) pharmacological studies to further evaluate in vitro anti-inflammatory activity. The interactions between the COX-2 active site and the most active compound 1 in comparison with the COX-2 inhibitor diclofenac are shown in Figure. Moreover, it should be noted that results predicted by molecular docking correlate quite well with that obtained in the in vitro assay. The selected "lead" compound 1 based on the in vitro screening results was also predicted to be the most active in the docking studies.
In contrast, the 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 for cyclooxygenase-1 during the docking experiment.
Evaluation of the anti-inflammatory activity in vivo. Carrageenan-induced paw edema is a T a b l e 1. Values of the Chemgauss 4 score of thiazolo [4,5-b] well-known animal model of acute inflammation, and is the most widely used in the search for new anti-inflammatory drugs. In vivo studies of novel thiazolo [4,5-b]pyridine-2-one derivatives were performed for anti-inflammatory activity. The results of the anti-inflammatory activity of the synthesized compounds and ibuprofen are shown in Table 2.
The synthesized compounds possess a range of anti-inflammatory activity -from its almost com-plete absence to a distinct anti-inflammatory effect. Evaluation indicated that 6 compounds (3, 4, 5, 7, 9, 10) showed no significant decrease in carrageenaninduced rat paw edema, as their inhibition rates were only 20.0-36.2%, as compared to the control group ( Table 2). The anti-inflammatory effect for compound 6 is approximately equivalent to that of the reference drug (ibuprofen). The anti-inflammatory effect for compounds 1, 2 and 8 resulted in inhibi- T a b l e 2. In vivo evaluation of anti-inflammatory effect of thiazolo [4,5-b] tion rates of 44.0-48.8%, indicating that these compounds were more potent than ibuprofen.

conclusion
The core thiazolo [4,5-b]pyridine heterocyclic system may be regarded as a promising scaffold for the development of effective anti-inflammatory drug candidates.