Synthesis, characterization and in vitro antimicrobial assessment of some novel 4H-1, 4-benzothiazines and their sulfone derivatives

Chemistry

The starting substituted 2-aminobenzenethiols 1a–d (Scheme 1) were synthesized by alkaline hydrolysis of corresponding 2-aminobenzothiazoles, which in turn were prepared by the cyclization of substituted phenylthiourea. The phenylthiourea derivatives were obtained by the reaction of ammonium thiocyanate with substituted anilines. A mixture of 2-aminobenzenethiols 1a–d and β-diketones/β-ketoesters 2a–d was heated under reflux conditions in DMSO, which led to condensation and oxidative cyclization. Oxidation of 2-aminobenzenethiols produced bis-(2-aminophenyl)disulfide 3, which in turn underwent cyclization to form 4H-1,4-benzothiazines 4a–e by scission of a sulfur-sulfur bond. This cleavage is due to high reactivity of the α-position of the enaminoketone system towards nucleophillic attack. Compounds 4a–e were converted into their corresponding sulfones 5a–e by treatment with 30% hydrogen peroxide in glacial acetic acid.

Scheme 1

Synthesis of 4H-1,4-benzothiazines 4a–e and their sulfone derivatives 5a–e.

Spectral data and elemental analysis support the structures of synthesized compounds. In the IR spectra of compounds 4a–e, a single peak is seen in the region of 3340–3310 cm^-1 due to NH stretching vibration. These compounds also exhibit a band at 1655–1605 cm^-1 due to C=O stretching vibrations. A slight shift towards higher frequencies is observed for sulfone derivatives 5a–e due to an increased electron-accepting ability of sulfones compared with the parent system. Compounds 5a–e exhibit intense peaks in the regions of 1385–1245 cm^-1, 1190–1145 cm^-1 and 585–545 cm^-1, which can be ascribed to vibrations of the sulfonyl group. The signals for C-S stretching vibrations in the region of 1065–1035 cm^-1 are also observed in the IR spectra of compounds 5a–e. The given structures of products 4a–e and 5a–e are also fully consistent with their ¹H NMR spectra. A multiplet in the region of δ 8.02–6.13 is due to the presence of aromatic protons in all compounds 4a–e and 5a–e. A singlet in the region δ 9.03–8.12 can be ascribed to the N-H function. The molecular ion peaks in the mass spectra of 4H-1,4-benzothiazines and their sulfone derivatives are fully consistent with their molecular weights. The base peak is for the ion R₄CO⁺, as predicted for the generation of the most stable cation.

Antimicrobial assessment

The in vitro antimicrobial assessment of synthesized compounds was carried out by using the agar well diffusion method for determination of AI and the broth microdilution method for determination of MIC against two strains of bacteria, Micromonospora sp. (Gram-positive) and Zymomonas mobilis (Gram-negative), and two strains of fungi, Aspergillus solani and Fusarium culmorum. Ampicillin sulfate and fluconazole were used as a standard antibacterial and antifungal drug, respectively. Compounds 4c and 5c show good antibacterial activity against the Gram-positive strain Micromonospora sp., whereas compounds 4d, 4e, 5d and 5e show excellent activity against the Gram-negative strain of subject bacteria Z. mobilis. Compound 5a exhibits good antifungal activity against A. solani and compound 5c is an excellent agent against F. culmorum. The remaining compounds show moderate antimicrobial activities. It should be noted that sulfone derivatives of 4H-1,4-benzothiazines exhibit better antimicrobial activities than their parent compounds. This finding can be explained by the presence of the electron-withdrawing group (-SO₂) in sulfones, which enhances biological activity. This effect of an electron-withdrawing group has been noted in the literature [16–18].

General method for synthesis of substituted 4H-1,4-benzothiazines 4a–e

β-Diketone/β-ketoester 2a–d (0.01 mol) was treated with a solution of 2-aminobenzenethiol 1a–d (0.01 mol) in 5 mL of DMSO and the resulting mixture was heated under reflux (190°C) for 20 min, then cooled and concentrated on a rotary evaporator. The residue was washed with petroleum ether and crystallized from methanol.

General method for synthesis of 4H-1,4-benzothiazine 1,1-dioxides (sulfones) 5a–e

A solution of 0.01 mol of 4H-1,4-benzothiazine in 20 mL glacial acetic acid was added to 30% hydrogen peroxide (5 mL) and then the mixture was heated under reflux for 15 min keeping the temperature between 50°C and 55°C. Another lot of 30% hydrogen peroxide (5 mL) was added after 15 min without heating. Then the mixture was heated under reflux (120°C) for an additional 4–5 h, concentrated under reduced pressure and the residue was treated with crushed ice. The resultant solid product 5 was filtered and crystallized from ethanol.

Antimicrobial assessment

MIC The broth microdilution method was used to determine the MICs (μg/mL) of the compounds in accordance with the NCCLS 1992 manual. The strains used for quality control were Micromonospora sp. MTCC 3296 (Gram-positive) and Z. mobilis MTCC 88 (Gram-negative) bacteria. Aspergillus solani MTCC 2101 and F. culmorum MTCC 349 were the reference strains for testing antifungal activities of the compounds. The standard antibacterial and antifungal drugs used were ampicillin sodium salt and fluconazole, respectively. DMSO was used for preparing solutions of test compounds and standard drugs. For preparing stock solution, each synthesized drug was diluted to obtain concentration of 1000 μg/mL. For primary screening, 500, 250 and 125 μg/mL concentrations of the synthesized drugs were taken. The active drugs found in this primary screening were further tested against all microorganisms in a second set of dilution in which the drugs were again diluted to obtain 100, 50, 25, 20, 15 μg/mL concentrations. The highest dilution showing at least 99% inhibition was recorded as MIC. Thus, the lowest concentration of each compound in the tube showing no growth (i.e., no turbidity) of inoculated bacteria/fungi was taken as MIC of that compound. Luria broth (Himedia) medium was used to carry out antibacterial activities of the bacterial strains. The reference strains of fungi were cultivated, at pH 6.9, in sabouraud dextrose agar (Himedia), with an inoculum of 10⁸ cfu/mL, by the spectrophotometric method. An aliquot (10 μL) of this prepared culture was added to each tube of the serial dilution and then incubated at 37°C for 24 h at 3.2602 × g (150 rpm) on a rotary shaker. After incubation, MIC values were recorded. The MICs of tested compounds in μg/mL against certain strains of bacteria and fungi are shown in Tables 1 and 2.

AI The AI of compounds was determined by the agar well diffusion method. In this method, nutrient agar and potato dextrose agar plates were swabbed with an 8-h-old broth culture of respective bacteria (Micromonospora sp. MTCC 3296 and Z. mobilis MTCC 88) and fungi (A. solani MTCC 2101 and F. culmorum MTCC 349). Wells were made on agar surface (6 mm in diameter and 2 cm apart) and punctured in the culture medium using sterile cork borers. The plates were then turned upside down. Stock solution of each compound was prepared at a concentration of 1 mg/mL in DMSO. The wells were filled with 100 μL of the compounds and one well was filled with 100 μL of DMSO which served as a control. The compounds were incubated at 37°C for 24 h in the case of bacterial activity and for 48 h at 25°C in the case of fungal activity. The plates were observed for the zone clearance around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in cm) including the well diameter. The zone of inhibition exhibited by compounds was then compared with the zone exhibited by the reference compounds (ampicillin sodium salt in the case of bacteria and fluconazole in the case of fungi) to find relative activity. The activity indices of tested compounds against certain bacteria and fungi were calculated as shown below and the results are tabulated in Tables 1 and 2.