A Novel Family of [1,4]Thiazino[2,3,4-ij]quinolin-4-ium Derivatives: Regioselective Synthesis Based on Unsaturated Heteroatom and Heterocyclic Compounds and Antibacterial Activity

A novel family of [1,4]thiazino[2,3,4-ij]quinolin-4-ium derivatives was synthesized by annulation reactions of 8-quinolinesulfenyl chloride with unsaturated heteroatom and heterocyclic compounds. It was found that the reactions with 4-pentenoic and 5-hexenoic acids, allyl chloride and bromide, allyl cyanate and vinyl heterocyclic compounds (N-vinyl pyrrolidin-2-one and 1-vinylimidazole) proceeded in a regioselective mode but with the opposite regiochemistry. The reactions with vinyl heterocyclic compounds included electrophilic addition of the sulfur atom of 8-quinolinesulfenyl chloride to the β-carbon atom of the vinyl group. In the case of other substrates, the annulation proceeded with the attachment of the sulfur atom to the α-carbon atom of the vinyl group. The antibacterial activity of novel water-soluble compounds against Enterococcus durans, Bacillus subtilis and Escherichia coli was evaluated. Compounds with high antibacterial activity were found.

A combination of the quinoline scaffold with condensed sulfur-containing heterocycles has proven a fruitful approach in the development of new drugs [6,7]. Valuable examples of such combinations include penicillin and cephalosporin antibiotics, as well as the fluoroquinolone antibiotics prulifloxacin and rufloxacin ( Figure 1). Levofloxacin and nadifloxacin represent antibiotics containing the quinoline scaffold condensed with sixmembered cyclic structures (Figure 1).
Compounds 4 and 5 are light yellow water-soluble powders with a melting point above 160 °C.
The reaction of 8-quinolinesulfenyl chloride 3 with allyl cyanate was very sluggish at room temperature in methylene chloride. However, carrying out the reaction of sulfenyl chloride 3 with allyl cyanate for 8 h in refluxing chloroform made it possible to obtain 2-cyanomethyl-2Н,3Н- [1,4]thiazino[2,3,4-ij]quinolin-4-ium chlorides 8 with a 96% yield (Scheme 5). Refluxing the reaction mixture in methylene chloride for 8 h led to product 8 with only a 67% yield. The involvement of substrates bearing potentially pharmacophoric heterocycles in annulation reactions is important in terms of the possible manifestation of biological activity. 1-Vinylimidazole and N-vinyl pyrrolidin-2-one, which contain a vinyl group bonded to a nitrogen atom, were involved in the annulation reactions with 8-quinolinesulfenyl chloride 3. The latter compound is an example of a heterocycle bearing a vinyl amide moiety in its structure.
Under the same conditions, the annulation reaction of 8-quinolinesulfenyl chloride 3 with N-vinyl pyrrolidin-2-one gave the annulation products with a 59% yield along with some by-products. It was found that this reaction proceeded more efficiently and selectively in the presence of potassium perchlorate. Under the same conditions, the annulation reaction of 8-quinolinesulfenyl chloride 3 with N-vinyl pyrrolidin-2-one gave the annulation products with a 59% yield along with some by-products. It was found that this reaction proceeded more efficiently and selectively in the presence of potassium perchlorate. Attempts were made to increase the yields by refluxing the reaction mixture in methylene chloride or chloroform. This made it possible to obtain products 9 and 10 with 90-94% yields; however, the selectivity of the reactions decreased, and compounds 9 and 10 were contaminated with by-products (6-10%), from which it was difficult to separate the target compounds.
The reactions with 4-pentenoic and 5-hexenoic acids, allylchloride, allylbromide and allyl cyanate included the electrophilic addition of the sulfur atom from sulfenyl chloride 3 to the α-carbon atom of the vinyl group ("anti-Markovnikov direction"), while the annulation reactions with N-vinyl pyrrolidin-2-one and 1-vinylimidazole proceeded with the attachment of the sulfur atom to the β-carbon atom of the vinyl group ("Markovnikov direction"). We presume that the reactions of sulfenyl chloride 3 with N-vinyl pyrrolidin-2-one and 1-vinylimidazole proceed via linear intermediates B (Scheme 7) which are stabilized by the nitrogen atom (the nitrogen atom's ability to stabilize adjacent carbocation is well known [42]). Attempts were made to increase the yields by refluxing the reaction mixture in methylene chloride or chloroform. This made it possible to obtain products 9 and 10 with 90-94% yields; however, the selectivity of the reactions decreased, and compounds 9 and 10 were contaminated with by-products (6-10%), from which it was difficult to separate the target compounds.
Vinyl ethers are promising substrates for annulation reactions due to the high reactivity of these compounds in electrophilic additions. The reactions of sulfenyl chloride 3 with ethyl vinyl and butyl vinyl ethers proceeded smoothly at room temperature in methylene chloride, producing 3-ethoxy-and 3-butoxy-2H,3H- [1,4]  Like the synthesis of products 9 and 10, the reactions of sulfenyl chloride 3 with ethyl vinyl and butyl vinyl ethers are believed to occur via linear intermediates (similar to intermediate B, Scheme 7), which are stabilized by the oxygen atom (the oxygen atom exhibits a strong ability to stabilize adjacent carbocation [53]).
Finally, based on the reaction of sulfenyl chloride 3 with cyclic vinyl ether, 2,3-dihydrofuran, we synthesized the condensed four-membered heterocycle 13, which Like the synthesis of products 9 and 10, the reactions of sulfenyl chloride 3 with ethyl vinyl and butyl vinyl ethers are believed to occur via linear intermediates (similar to intermediate B, Scheme 7), which are stabilized by the oxygen atom (the oxygen atom exhibits a strong ability to stabilize adjacent carbocation [53]).
Finally, based on the reaction of sulfenyl chloride 3 with cyclic vinyl ether, 2,3dihydrofuran, we synthesized the condensed four-membered heterocycle 13, which is of interest for evaluation of antibacterial activity and comparison with the antibacterial properties of products 11 and 12, obtained from ethyl vinyl and butyl vinyl ethers. The reactions of sulfenyl chloride 3 with 2,3-dihydrofuran was carried out in the presence of an equimolar amount of KClO 4 at room temperature in methylene chloride, leading to perchlorate 13 with a 72% yield (Scheme 9).  Similarly to the reactions with ethyl vinyl and butyl vinyl ethers (Scheme 8), syn thesis of compound 13 was regioselective and the sulfur atom of sulfenyl chloride bonded to the β-carbon atom of the vinyloxy group.
The antibacterial activity of the synthesized compounds was evaluated. The min mal inhibitory concentration (MIC) was determined using the broth standard microdilu tion method [54].
Compounds 1, 4-13 were tested in vitro for antibacterial activity against bacteria strains of gram-positive Enterococcus durans B-603, Bacillus subtilis B-406 an gram-negative Escherichia coli B-1238 (the bacterial strains were taken from th All-Russian Collection of Microorganisms) and the obtained results were compared t the activity of standard aminoglycoside antibiotic gentamicin (the minimal inhibitor concentrations are 25, 50 and 100 μg/mL against E. durans, B. subtilis and E. coli, respe tively). The obtained results are presented in the Table 1. Similarly to the reactions with ethyl vinyl and butyl vinyl ethers (Scheme 8), synthesis of compound 13 was regioselective and the sulfur atom of sulfenyl chloride 3 bonded to the β-carbon atom of the vinyloxy group.
The antibacterial activity of the synthesized compounds was evaluated. The minimal inhibitory concentration (MIC) was determined using the broth standard microdilution method [54].
Compounds 1, 4-13 were tested in vitro for antibacterial activity against bacterial strains of gram-positive Enterococcus durans B-603, Bacillus subtilis B-406 and gram-negative Escherichia coli B-1238 (the bacterial strains were taken from the All-Russian Collection of Microorganisms) and the obtained results were compared to the activity of standard aminoglycoside antibiotic gentamicin (the minimal inhibitory concentrations are 25, 50 and 100 µg/mL against E. durans, B. subtilis and E. coli, respectively). The obtained results are presented in the Table 1.
The activities of compounds 4 and 5, which differ only in one CH 2 group, are significantly different. Compound 5, with its longer carbon chain, exhibited considerably higher activity against gram-positive E. durans and B. subtilis and is superior to antibiotic gentamicin in this respect (Table 1).                 The products with the opposite regiochemistry show the characteristic signals of carbon atoms bonded with a charged nitrogen (N + ) atom and a sulfur atom. The number of protons (one or two) bonded to the carbon atoms adjacent to the charged nitrogen atom and to the sulfur atom is important (the number of protons is determined by NMR experiments). For example, the CHS moiety and the CH2N + methylene group manifested themselves in the regions of 32-43 ppm and 58-64 ppm, respectively, in the 13 C-NMR spectra of compounds 4-8 (the products derived from anti-Markovnikov addition of the sulfur electrophile to the double bond). Signals of the one-proton-containing OCHN + moiety were observed in the downfield region of 91-92 ppm in the 13 C-NMR spectra of compounds 11-13 (the products derived from Markovnikov addition of the sulfur electrophile to the double bond).

General Information
The 1 H (400.1 MHz) and 13 C (100.6 MHz) NMR spectra were recorded on a Bruker DPX-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) in 2-5% solution in D2O, DMSO-d6, methanol-d4 or acetone-d6. 1 H and 13 C chemical shifts (δ) were reported in parts per million (ppm), relative to tetramethylsilane (external) or to the residual solvent peaks of D2O (δ = 4.79), acetone-d6 (δ = 2.05 and 29.84 ppm), methanol-d4 (δ = 3.31 and 49.0 ppm) and DMSO-d6 (δ = 2.50 and 39.52 ppm for 1 H and 13 C NMR, respectively). The term "quino" in spectral data indicates belonging to the quinoline ring. The elemental analysis was performed on a Thermo Scientific FLASH 2000 Organic Elemental Analyzer (Thermo Fisher Scientific Inc., Milan, Italy). Melting points were determined on a Kofler Hot-Stage Microscope PolyTherm A apparatus (Wagner & Munz GmbH, München, Germany). Absolute solvents were used in the reactions. [1,4]thiazino [2,3,4-ij]quinolin-4-ium chloride (4). A solution of sulfuryl chloride (0.076 g, 0.56 mmol) in chloroform (10 mL) was added dropwise to a solution of di(8-quinolinyl) disulfide (0.180 g, 0.56 mmol) in chloroform (10 mL), and the mixture was stirred for 10 min at room temperature. A solution of pentenoic acid (0.112 g, 1.12 mmol) in chloroform (10 mL) was added dropwise, and the reaction mixture stirred for 1 h at room temperature and 8 h at reflux temperature. After cooling in the refrigerator, the formed precipitate was filtered off and dried in a vacuum, producing the product (0.232 g, 70% yield) as a yellow powder, mp 170-172 °C. 1  Compounds 6 and 7 differ only in the halogen atom. Bromo-containing compound 7 was 40 times more effective than its chlorine analogue 6 against E. durans. However, product 6 was the most effective among the obtained compounds against gram-negative bacteria E. coli. Silicon-containing product 1 and compound 8 showed low activity. Compound 9 exhibited average activity against all tested bacteria ( Table 1).
The highest activity was shown by product 10 (obtained from N-vinyl pyrrolidin-2one), which significantly exceeded the activity of gentamicin and all obtained compounds against gram-positive bacteria and was more than a hundred times superior to this antibiotic against B. subtilis ( Table 1).
The structural assignments of synthesized compounds were made using 1 H and 13 C-NMR spectroscopy, including two-dimensional experiments (Supplementary Materials containing examples of NMR spectra are available online), and confirmed by elemental analysis.
The products with the opposite regiochemistry show the characteristic signals of carbon atoms bonded with a charged nitrogen (N + ) atom and a sulfur atom. The number of protons (one or two) bonded to the carbon atoms adjacent to the charged nitrogen atom and to the sulfur atom is important (the number of protons is determined by NMR experiments). For example, the CHS moiety and the CH 2 N + methylene group manifested themselves in the regions of 32-43 ppm and 58-64 ppm, respectively, in the 13 C-NMR spectra of compounds 4-8 (the products derived from anti-Markovnikov addition of the sulfur electrophile to the double bond). Signals of the one-proton-containing OCHN + moiety were observed in the downfield region of 91-92 ppm in the 13 C-NMR spectra of compounds 11-13 (the products derived from Markovnikov addition of the sulfur electrophile to the double bond).

Synthesis of Compounds 4-8
2-(3-Carboxyethyl)-2H,3H- [1,4]thiazino [2,3,4-ij]quinolin-4-ium chloride (4). A solution of sulfuryl chloride (0.076 g, 0.56 mmol) in chloroform (10 mL) was added dropwise to a solution of di(8-quinolinyl) disulfide (0.180 g, 0.56 mmol) in chloroform (10 mL), and the mixture was stirred for 10 min at room temperature. A solution of pentenoic acid (0.112 g, 1.12 mmol) in chloroform (10 mL) was added dropwise, and the reaction mixture stirred for 1 h at room temperature and 8 h at reflux temperature. After cooling in the refrigerator, the formed precipitate was filtered off and dried in a vacuum, producing the product (0.232 g, 70% yield) as a yellow powder, mp 170-172 • C. 1 (5). A solution of sulfuryl chloride (0.087 g, 0.64 mmol) in chloroform (10 mL) was added dropwise to a solution of di(8-quinolinyl) disulfide (0.206 g, 0.64 mmol) in chloroform (10 mL), and the mixture was stirred for 10 min at room temperature. A solution of hexenoic acid (0.147 g, 1.28 mmol) in chloroform (10 mL) was added dropwise, and the reaction mixture stirred for 1 h at room temperature and 8 h at reflux temperature. After cooling in the refrigerator, the formed precipitate was filtered off and dried in a vacuum, producing the product (0.286 g, 72% yield) as a yellow powder, mp 161-162 • C. 1 (7). A solution of sulfuryl chloride (0.082 g, 0.60 mmol) in chloroform (10 mL) was added dropwise to a solution of di(8-quinolinyl) disulfide (0.194 g, 0.60 mmol) in chloroform (10 mL), and the mixture was stirred for 10 min at room temperature. A solution of allyl bromide (0.147 g, 1.2 mmol) in chloroform (10 mL) was added dropwise, and the reaction mixture stirred for 1 h at room temperature and 8 h at reflux temperature and 16 h at room temperature. The mixture was filtered and the solvent was removed by rotary evaporator. The residue was dried in a vacuum, producing the product (0.342 g, 90% yield) as a yellow powder, mp 162-164 • C. 2-(Cyanomethyl)-2H,3H- [1,4]thiazino[2,3,4-ij]quinolin-4-ium chloride (8). A solution of sulfuryl chloride (0.059 g, 0.44 mmol) in chloroform (10 mL) was added dropwise to a solution of di(8-quinolinyl) disulfide (0.140 g, 0.44 mmol) in chloroform (10 mL), and the mixture was stirred for 10 min at room temperature. A solution of allyl cyanide (0.059 g, 0.88 mmol) in chloroform (10 mL) was added dropwise, and the reaction mixture stirred for 1 h at room temperature and 8 h at reflux temperature. After cooling in the refrigerator, the formed precipitate was filtered off and dried in a vacuum, producing the product (0.223 g, 96% yield) as a yellow powder, mp 183-185 • C.