Convenient Synthesis of 4-pyridinyloxy-Modified imidazo[2,1- b ][1,3]thiazines as Potential Anti-inflammatory Agents

: Novel imidazo[2,1- b ][1,3]thiazine derivatives modified with 4-pyridinyloxyl moiety, as potential anti-inflammatory agents are described. Synthetic approach to the preparation of (6-((pyridin-4-yl)oxy)-6,7-dihydro-5 H -imidazo[2,1- b ][1,3]thiazines 3a-f and their benzoanelated analogues 3g-j is based on the interaction of substituted 4-fluoropyridines with 3-hydroxy(benzo)imidazo[2,1-b ][1,3]thiazines. The nucleophilic substitution reaction of the fluorine atom occurs selectively at position 4 of the pyridine ring. The drug-like properties of the first synthesized (4-pyridinyloxy)imidazo[2,1- b ][1,3]thiazines were predicted in silico using SwissADME. Anti-inflammatory activity was studied in vivo using hind paw edema in white rats (carrageenan test). Compounds with satisfactory drug-like and pharmacological properties have been identified as promising for further structure optimization and in-depth research.

Recently [18], we developed an effective method for the synthesis of a series of new azolo-azine-type derivatives-6-[(pyridin-2-yl)oxy] -6,7-dihydro-5H-imidazo [2,1-b] [1,3] thiazines and their benzoanelated analogs, which testified to the prospects for the search for bioactive substances by structural functionalization of the imidazo [2,1-b]thiazine scaffold by pyridine fragment. Indeed, synthesized for the first time, hybrid imidazo-thiazine-pyridine compounds showed moderate antibacterial, antifungal, and anti-inflammatory activity in experimental in vitro and in vivo studies [18]. It seems quite probable that this result is largely due to the diverse biological effect of the pyridine scaffold [19][20][21]. In particular, substituted pyridine-3sulfonamides I ( Figure 2) show a broad spectrum of inhibition of cancer cell growth in 26 lines [22], good activity, and selectivity for subpanels of leukemia colon cancer, and melanoma [23]. In turn, pyridinyl-containing vinyl ketone II inhibits the proliferation of human melanoma cells [24]. In addition, N- (1,2,4-triazol-3-yl)pyridine-3-sulfonamides III show greater antifungal efficacy than fluconazole [25], and pyridyl-1,4-dihydropyridines IV possess antihypertensive properties at an equal level to the nicardipine [26]. Polysubstituted pyridine derivatives are inhibitors of trypsin [27], β-lactamase [28] and phosphodiesterase (PDE2A) [29]. Given that the construction of molecules containing several pharmacophores is one of the most effective approaches to the search for bioactive substances, we thought it appropriate to develop a selective method for the synthesis of new derivatives of imidazo [2,1b] [1,3]thiazines modified in thiazine nucleus with 4-pyridinyloxy fragments and evaluate their drug-like and anti-inflammatory properties. In this context, the presented work is a development of previously published results [18], which concerned the synthesis and some biological properties of 2-pyridinyloxy-containing (benzo)imidazo [2,1-b] [1,3]thiazines.

Materials.
All reagents were chemically pure and used without further purification. The solvents were purified according to standard procedures [30].

Chemistry.
Melting points were measured on a Kofler melting point-device and are uncorrected. IR spectra were recorded on Bruker Vertex 70 FT-IR spectrometer for samples in KBr pellets. 1 H NMR spectra were acquired in pulsed Fourier transform mode on a Varian VXR-400 spectrometer (400 MHz), while 13 CNMR spectra were acquired on a Bruker Avance DRX-500 spectrometer (125 MHz) using DMSO-d6 as solvent. Mass spectra were recorded on an Agilent LC/MSD SL chromatograph equipped with Zorbax SB-C18 column (4.6x15mm), particle size 1.8 µm (PN 82(c)75-932), solvent DMSO, electrospray ionization at atmospheric pressure. Elemental analysis was performed on a PerkinElmer 2400 CHN Analyzer. The individuality of the obtained compounds was monitored by TLC on Silutol UV-254 plates.

Anti-inflammatory (antiexudative) 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 the use and care of animals in the declaration of Helsinki (National Research Council, 2011). The Animal Ethics Committee approved the experiment design and study protocol of the Danylo Halytsky Lviv National Medical University, protocol No.10, March 17, 2021. The carrageenan-induced hind paw edema was produced by Winter et al. [31]. The compounds synthesized 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 edema, was calculated using the equation, and was given in percentage: where, ΔVcontrol and ΔVexperimentthe mean values of the volume difference for control and experimental animals hinds, respectively.

Assessment of liver function.
The serum collected from the albino rats was used to estimate biochemical parameters to determine the functional state of the liver. The levels of total alkaline phosphatase (ALP), gamma-glutamyltransferase (γ-GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were estimated photometrically according to the reported methods using CORMAY ACCENT-200 automatic analyzers (PZ Cormay, Poland).

Ulcerogenic activity estimation.
All animals were sacrificed under deep anesthesia 6 hours after drug treatment, and then their stomachs were removed, opened along the great curvature, and rinsed with a saline solution of 0.9%. The gastric mucosa was examined using a magnifying glass (2X) to assess the incidence of redness and spot ulcers. The mucosal damage was evaluated according to the following score: 0 -no visible damage; 1 -the presence of edema or hemorrhages, 1-3 small ulcers; 2 -several (more than 3) small ulcers or 1 ulcer of considerable size; 3 -ulcer of considerable size (diameter up to 4 mm); 4 -several large ulcers; 5 -breakthrough ulcer. The gastric mucosal ulceration score was calculated by the difference between the mean score of each treated group and the control group's mean score.

Statistical analysis.
All data were processed using the statistical package Statistica 10.0 (Statsoft/Dell, Tulsa, OK, USA). The descriptive statistics of the data in the tables include mean ± standard error of the mean (SEM) or mean ± standard deviation. Significance was assessed by using the one-way ANOVA followed by -test. Values were considered statistically significant when value was less than 0,05.

Chemistry.
For the synthesis of target structurally modified imidazo [2,1-b] [1,3]thiazines with 4pyridinyloxy fragments, we have proposed an approach based on pyridinylation of available 3hydroxyimidazo[2,1-b] [1,3]thiazines 1a,b [32] and their benzoanalogue 1c [12] with 4-fluorocontaining pyridines 2a-d. It was found that, despite the bi-and polyfunctional electrophilic nature of pyridines 2a-d, this reaction is characterized by high regioselectivity and is realized exclusively as a nucleophilic substitution of the fluorine atom at position 4 of the pyridine ring on oxyimidazo[2,1-b] [1,3]thiazino fragment with the formation of 6-[(pyridin-4-yl)oxy]-6,7dihydro-5H-imidazo[2,1-b][1,3]thiazines 3a-j (Scheme 1). The LC-MS method did not detect nitro substitution products and chlorine or iodine atoms in the reaction mixtures, most likely due to the stronger polarization of the C-F bond by the sp 2 -hybridized pyridine nitrogen atom. The selection of experimental conditions revealed that the optimal for the reaction is the use of DMF as a solvent and NaH as a base. Compounds 3a, b, d, f, h, j are obtained in 58-73% yields by carrying out the reaction at room temperature for 24 hours. Instead, synthesizing compounds 3c, e, g, i containing 3-nitropyridinyl-and 3,5-dichloropyridinyl fragments require heating the reaction mixtures at 80 ° C for 5 hours. The structure of the synthesized compounds was confirmed by 1H NMR, 13C NMR, and LC-MS spectra, which are presented in the experimental part. In particular, in the 1H NMR spectra of all imidazothiazines 3a-j, the pyridine cycle proton signals are presented, and derivatives 3a, b are easily identified in the range of 8.53-6.86 ppm, and their diphenyl 3c-f and benzo analogs, 3g-j are overlapped with signals of the phenyl-group protons.
The control of the reaction process and products formation was monitored by TLC. The compounds' structure characterization and yield are presented in Table 1.

In silico evaluation of drug-likeness properties.
The drug-likeness properties of the derivatives 3a-j were determined based on Lipinski and Veber rules and evaluated in silico using the SwissAdme of the Swiss Institute of Bioinformatics website [33] (Table 2). Accordingly, with obtained data, almost all derivatives (except 3e and 3f) correspond to the Lipinski and Veber rules and possess satisfactory pharmacokinetic parameters with a high level of predicted gastrointestinal absorption.

Investigation of anti-inflammatory (anti-exudative) activity.
The anti-inflammatory (anti-exudative) activity of all synthesized compounds 3 a-j was investigated in the in vivo carrageenin model of the total edema of hind paws of albino rats [31]. The study results are presented in Table 3. The synthesized compounds 3 a-j were mostly active under carrageenin-induced paw edema conditions. The inhibition index was observed in the range of 14.1 to 41.6 %. From this point of view, the "structureanti-inflammatory activity" derivatives 3 a-j with unsubstituted imidazole ring in the imidazo[2,1-b] [1,3]thiazine core are characterized by a total higher activity level. Such data correlate with our early obtained results and are in accordance with in silico predicted drug-like and pharmacokinetic properties. It should be noted that monochlorosubstituted derivatives 3а, 3h were found to be the most active, whereas the introduction of the additional chlorine, iodine atoms, or nitro-group led to activity decreasing.
The impact on the function of liver enzymes was studied for the most active derivatives 3a and 3h. Administration of tested derivatives 3a and 3h and reference drugs do not negatively impact liver function (Table 4). The derivatives 3a and 3h were evaluated for ulcerogenic activity after application at a 50 mg/kg dose in rats. The results were compared with an intact control group and the diclofenac sodium group ( Table 5). As a result, diclofenac sodium showed significant ulcerogenic risk with a high ulceration score. The tested compounds didn't show any ulcerogenic activity.

Funding
This research received no external funding.