Efficient and convenient synthesis, characterization, and antimicrobial evaluation of some new tetracyclic 1,4-benzothiazines

ABSTRACT In the present study, 20 new tetracyclic 1,4-benzothiazines (4a–4 t) were conveniently synthesized in good yields and characterized by different spectral and physical techniques. The in vitro antimicrobial evaluation of the synthesized benzothiazine derivatives was performed by serial dilution against two Gram-positive bacteria [Bacillus subtilis (MTCC 441) and Staphylococcus epidermidis (MTCC 6880)], two Gram-negative bacteria [Escherichia coli (MTCC 1652) and Pseudomonas aeruginosa (MTCC 424)], and two fungal strains [Candida albicans (MTCC 227) and Aspergillus niger (MTCC 8189)]. The derivatives 4 l and 4 t were found to be more potent than standard drug, i.e., fluconazole, against A. niger and C. albicans, respectively. GRAPHICAL ABSTRACT

The structures of all the newly synthesized 1,4-benzothiazines (4a-4 t) were elucidated on the basis of spectral (infrared [IR], 1 H NMR, 13  , and mass) as well as analytical results. The IR spectra of all these compounds (4a-4 t) displayed the characteristic absorptions due to C = O stretching and C = N stretching in the regions at 1676-1697 cm À 1 and 1645-1651 cm À 1 , respectively. The 1 H NMR spectra of compounds (4a-4 t), in each case, exhibited a one-proton doublet in the region at δ 6.46-6.58 (J ¼ 7.92-8.08 Hz), which was undoubtedly assigned to C 4 -H. The compounds with substitution at C 8 displayed a one-proton doublet in the region at δ 6.60-7.21 safely assignable to C 9 -H, with coupling constant (J) ranging from 1.36-2.88 Hz, which is due to meta coupling. The one-proton doublet (J ¼ 7.44-7.68 Hz) exhibited in the region at δ 7.91-7.97 was easily assigned to C 1 -H. The most characteristic feature of 1 H NMR spectra of 1,4-benzothiazines (4a-4 t) was a one-proton doublet (J ¼ 8.  in the region at δ 9.04-9.18 due to C 6 -H. The signals due to the remaining aliphatic and aromatic protons were observed in their expected regions. The salient feature of 13 C NMR spectra of 1,4-benzothiazines (4a-4 t) was the signal observed in the most downfield region at δ 164.80-165.22, which was indubitably assigned to C 11 . Further, it is worth mentioning here that with the variation of R, the change in chemical shift value of C 8 signal was observed. The signals due to the remaining aliphatic and aromatic carbons were observed in the expected regions. Further, the mass spectral data and elemental analysis results of 1,4-benzothiazines (4a-4 t) were also found in good agreement with their molecular formulas. serial dilution technique. [45] Minimum inhibitory concentrations (MIC) were expressed in terms of µmol/mL. Ciprofloxacin and Fluconazole ( Fig. 1) were used as reference drugs for antibacterial and antifungal evaluations, respectively. The results of antibacterial evaluation summarized in Table 1 and Fig. 2 revealed that the derivatives 4a, 4f, 4i, and 4n against B. subtilis; 4a, 4d, 4f, 4 g, 4i, 4k, 4 l, 4o, and 4p against S. epidermidis; 4a, 4d, 4f, 4 g, 4i, 4k, 4n, 4o, 4s, and 4t against E. coli; and 4 g and 4h against P. aeruginosa were moderately active as compared to the standard reference. However, all the tested derivatives were found to exhibit less inhibitory activity as compared to reference drug. Further, the results of antifungal evaluation presented in Table 2 and Fig. 3 revealed that derivatives 4a, 4f, 4j, and 4p exhibited moderate activity against C. albicans as compared to the reference drug Fluconazole. Similarly, compounds 4h and 4r also possessed appreciable antifungal activity against A. niger as compared to the reference drug. Interestingly, the compounds 4t against C. albicans and 4l against A. niger were found to be more potent than the standard drug.  Hence, 4 t and 4 l can be used as potential antifungal agents for further drug development. Moreover, comparison of antibacterial and antifungal evaluation results reveals that the antifungal activities are more prolific than antibacterial activities.

In vitro antimicrobial evaluation
From these antimicrobial results, the following structure-activity relationships (SAR) may be inferred: 1. The unsubstitution at C 8 of 1,4-benzothiazine ring improved the antimicrobial efficacy against S. epidermidis, E. coli, and C. albicans. 2. The presence of electron-withdrawing groups such as F at C 4' of the C 10a -phenyl ring and Br at C 8 and/or C 4' positions enhanced the antimicrobial activity against all the tested microbial strains except P. aeruginosa.  3. In most of the cases, the presence of electron-donating ethyl group at C 4 0 of the C 10a -phenyl ring increased the antimicrobial potency against all the tested microbial strains. 4. In some cases, the presence of electron-withdrawing NO 2 at C 4 0 of the C 10a -phenyl ring improved the antimicrobial activity against S. epidermidis, B. subtilis, E. coli, and C. albicans. 5. In contrast to the previous results, the presence of electron-withdrawing groups such as NO 2 , F, and Br at C 4' of the C 10a -phenyl ring lessen the antibacterial activity against P. aeruginosa. These results lead to the conclusion that there are different structural requirements for a compound to be effective against different microbial strains. However, no general trend toward a structure-activity relationship (SAR) has been established for antimicrobial activity of the tested 1,4-benzotiazine derivatives (4).

Conclusion
Conclusively, we have accomplished the synthesis of a series of 20 new tetracyclic 1,4benzothiazines (4a-4 t) through an efficient chemical transformation. All the synthesized 1,4-benzothiazines (4a-4 t) have been investigated for their in vitro antimicrobial activity. Most of the tested compounds exhibited moderate to good activity towards Gram-positive and Gram-negative bacterial strains as well as the fungal strains. Among the target compounds, 4 l and 4 t were found to be more potent than standard drug against A. niger and C. albicans, respectively, which could be promising lead candidates for further drug discovery against fungal infections. Further work in this direction is continuing in our laboratory.

Experimental
Commercial reagents were utilized as received from suppliers without additional purification. 2-Aminothiophenol was purchased from Sigma-Aldrich. All the synthesized compounds were characterized by 1 H NMR, 13   chromatography (TLC) was used for monitoring the progress of the reaction and ascertaining the purity of the synthesized compounds on precoated TLC plates (Merck Keiselgel F 254 ) using a hexane-ethyl acetate solvent system of different polarities, and visualization was achieved by exposure to UV light. Columns were packed as slurry of silica gel (60-120 mesh) in hexane. Initially, compounds were adsorbed on silica gel in an appropriate solvent and then loaded on column as slurry in hexane. The FTIR spectra were scanned on IR Affinity-1 FTIR (Shimadzu) spectrophotometer in KBr and wave numbers (ν) are reported in cm À 1 . Nuclear magnetic resonance spectra ( 1 H at 400 MHz and 13 C at 100 MHz) were recorded on a 400-MHz Bruker Avance-III spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS) as internal standard. DEPT and 2D-NMR viz. COSY, HSQC, and HMBC spectra of compound 4b were also recorded on a 400-MHz Bruker Avance-III spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) and coupling constants (J) are expressed in hertz (Hz). Multiplicities in NMR signals are designated as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quartet), and m (multiplet, for unresolved signals). Mass spectra were scanned on a Waters quadrupole detector (TDQ), Waters GTOF micromass spectrometer, and Agilent 6410B Triple Quard LCMS spectrometer. Microanalyses were performed on a Thermo Scientific FLASH-2000 CHN analyzer. All the synthesized compounds were in good agreement with the elemental analysis. Analytical results for C, H, and N were found to be within �0.4% of the theoretical values. Nomenclature of the compounds was assigned with the help of Chem Draw Ultra 12.0.

Spectral data
Orange solid, yield 85%; mp 250-252°C; IR (KBr, cm  [45] Initially, stock solutions were prepared by dissolving weighed amounts of synthesized compounds in dimethylsulfoxide (DMSO; 1.0 mg of the test compound in 10 mL DMSO) to get a final concentration of 100 µg/mL. Fresh cultures were obtained by inoculation of respective microorganisms in suitable media (nutrient broth for bacterial strains and potato dextrose broth for fungal strains), followed by incubation at 37 � 1°C for 24 h (all bacteria), 25 � 1°C for 7 days (A. niger), and 37 � 1°C for 48 h (C. albicans). The stock solutions of the test compounds were then serially diluted in test tubes containing 1 mL of sterile medium to get the concentrations of 50-0.39 µg/mL. Then 100 µL of the respective microorganism in sterile saline was inoculated to different dilutions of test compounds (each dilution in triplicate). The inoculated test tubes were incubated at 37 � 1°C for 24 h (bacteria), 25 � 1°C for 7 days (A. niger), and 37 � 1°C for 48 h (C. albicans). Ciprofloxacin and Fluconazole were used as standard antibacterial and antifungal drugs, respectively, which were also assessed under similar conditions for comparison with the tested compounds. After incubation, microbial growth was monitored visually and spectrophotometrically and the results were recorded in terms of minimum inhibitory concentration (MIC, µmol/mL). The data for the antibacterial activity are presented in Table 1 and Fig. 2, and results of antifungal activity are depicted in Table 2 and Fig. 3.