Synthetic Approach to Diversified Imidazo[2,1- b ][1,3]thiazines and Its Evaluation as Non-Steroidal Anti-Inflammatory Agents

: The present work is devoted to the synthesis of imidazo[2,1- b ][1,3]thiazine derivatives as possible anti-inflammatory agents. The synthetic approach to (2-pyridinyloxy)substituted imid-azo[2,1- b ][1,3]thiazines based on the interaction of the polysubstituted 2-chloropyridines with 3-hydroxy-imidazo[2,1- b ][1,3]thiazines was proposed. Selective nucleophilic substitution in position 2 of pyridine ring was observed in the mentioned reaction. The synthesized (2-pyridinyloxy)substi-tuted imidazo[2,1- b ][1,3]thiazines drug-like properties were studied in silico using SwissADME and anti-inflammatory activity in carrageenan test in vivo. Hit-compounds with satisfactory drug-like and pharmacological features were identified as promising objects for futhercoming structure opti-mization and in-depth studies.

Taking into account that synthesis of the hybrid molecules containing two or more pharmacophores is a promising and interesting approach to in the design of potential pharmacological active small molecules, it seemed to be interesting to work out the straightforward and convenient protocol for the synthesis of new hybrid molecules containing diversified imidazo [2,1-b] [1,3]thiazine scaffolds linked with potential pharmacophore-pyridine ring and evaluate their drug-like and anti-inflammatory properties.

General Information
Melting points were measured in open capillary tubes on a BÜ CHI B-545 melting point apparatus (BÜ CHI Labortechnik AG, Flawil, Switzerland) and are uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer (PerkinElmer, Waltham, MA, USA) and were within ±0.4% of the theoretical values. The 400 MHz-1 H and 126 MHz-13 C spectra were recorded on Varian Unity Plus 400 (400 MHz) spectrometer (Varian Inc., Paulo Alto, CA, USA). All spectra were recorded at room temperature except where indicated otherwise and were referenced internally to solvent reference frequencies. Chemical shifts (δ) are quoted in ppm and coupling constants (J) are reported in Hz. LC-MS spectra were obtained on a Finnigan MAT INCOS-50 (Thermo Finnigan LLC, San Jose, CA, USA). The reaction mixture was monitored by thin layer chromatography (TLC) using commercial glass-backed TLC plates (Merck Kieselgel 60 F254). Solvents and reagents that are commercially available were used without further purification. The 3-hydroxy-imidazo[2,1-b] [1,3]thiazines 2a-c were prepared using the similar protocol described in [5].

Anti-Inflammatory (Anti-Exudative) Activity
The male albino rats weighing 180-220 g were used for anti-exudative activity studying. The animals were treated humanely throughout the study period adhering to the guideline for use and care of animals in declaration of Helsinki (National Research Council, 2011). The experiment design and study protocol were approved by the Animal Ethics Committee of the Danylo Halytsky Lviv National Medical University, Lviv, Ukraine, protocol No.10, 17 March 2021. The carrageenin-induced hind paw edema was produced by the method of Winter et al. [14]. The synthesized compounds were intraperitoneally injected in a dose 50 mg/kg (in saline solution with one drop of Tween-80™). Diclofenac (tablets "Diclofenac sodium", "Zdorovja narodu", Ukraine) in dose 8 mg/kg was used as reference drug. The antiexudative activity (inflammation inhibition) was expressed as a decrease of rats paw oedema, was calculated using the equation and was given in percentage: where, ΔVcontrol and ΔVexperiment-the mean values of the volume difference for control and experimental animals hinds respectively.

Results and Discussion
Used in the present work synthetic approach is based on the utilization of structuremodified imidazolinthiones as starting building block for the formation of imidazo [2,1b] [1,3]thiazine core. The interaction of the last ones in the soft conditions with epichlorohydrin lead to the key 3-hydroxy-imidazo[2,1-b] [1,3]thiazines 2a-c [5]. The various polysubstituted 2-chloropyridines were studied in the alkylation reaction with early synthesized compounds 2a-c. As results, the target (2-pyridinyloxy)substituted imidazo [2,1b] [1,3]thiazines 3a-m were obtained with satisfied yields (in the presence of equimolar amounts of 60% sodium hydride in anhydrous DMF medium) at room temperature and the selective nucleophilic substitution in position 2 of pyridine ring was observed. The control of reaction process and products formation was monitored by TLC. The compounds' structure characterization and yield are presented in the Table 1. Table 1. Structure characterization and yeilds of synthesized compounds 3a-m. All tested compounds comply with Lipinski's rules of five and Veber's rules, except, derivatives 3h and 3i for which calculated MlogP values were higher (4.69 and 4.28 accordingly) than limited for Mlog P parameter (accepted ≤ 4.15) in line with the Lipinski's rules.

Study of Anti-Inflammatory (Anti-Exudative) Activity of Synthesized Compounds 3a-m
The anti-inflammatory (anti-exudative) activity of all synthesized compounds 3a-m was investigated on the in vivo carrageenin model of the total edema of hind paws of albino rats [14]. The study results are presented in Table 3. Table 3. In vivo anti-inflammatory activity of compounds 3a-m on carrageenin-induced paw edema in white rats (intraperitoneally use; doses: carrageenin 1%, 0.1 mL; Diclofenac sodium-8 mg/kg, tested compounds-50 mg/kg; M ± m; n = 6 in each group). The synthesized compounds 3a-m possess different levels of anti-inflammatory activity (inhibition index was in the range of 3.7 to 39.1%). From the point of view the "structure-anti-inflammatory activity" derivatives 3a-d with unsubstituted imidazole ring in the imidazo[2,1-b] [1,3]thiazine core are characterized with total higher activity level. The compound 3c containing cyano-group in the pyridine ring was the most active among derivatives 3a-d, whereas the change of cyano-group on chlorine or threefluormethylgroup led to to insignificant activity decrease. Derivative 3l was found the most active inside the tested group with inflammation inhibition value of 39.1% what is only 15.5% less compare to the same data for reference-drug diclofenac.

Data Availability Statement:
The data presented in this study are available in this article.