Synthesis, spectral studies, antioxidant and antibacterial evaluation of aromatic nitro and halogenated tetradentate Schiff bases

Herein, we report the synthesis, characterization, and biological properties of eleven (3a-3k) novel Schiff bases. The spectral data of FT-IR, 1H NMR, 13C NMR, and LC-MS are associated with these synthesized compounds. From the FT-IR analysis, we confirmed the azomethine (-C=N-) group and from 1H NMR data, the phenolic –OH proton is appeared at range δ 13.92–14.09ppm due to hydrogen bonding. The LC-MS analysis agreed with molecular ion peaks of synthesized Schiff bases. To evaluate the antibacterial activity of newly synthesized compounds were screened against b. licheniformis, b. species, e. coli, and s. aureus. Furthermore, the antioxidant activity was investigated by two methods 2,2-diphenyl-1-picryl hydrazyl (DPPH) and hydroxyl radical scavenging methods. The (-NO2,-Cl,-Br,-I) substituted compounds have shown good antibacterial activity against tested organisms. Also, these compounds were exhibited higher antioxidant activity by given methods.


Chemical material and instrumentation
For the experimental work, the chemicals were purchased by aura, spectrochem & TCI and without further purification. In the laboratory, thin layer chromatography (TLC) took by 0.25-mm e merck gel Plates (60F-254). Synthesized compounds were dissolved in a minimum amount of acetic acid, spotted on the given TLC plate, and ran through a solution of ethyl acetate and benzene. The melting point determination of compounds was done by digital apparatus koefler banc. The elemental analysis of the synthesized compounds was carried out through perkinelmer 240 elemental analyzer. The structure of unknown compounds was agreed upon by different spectral characterization. FT-IR spectrometer was recorded KBr pellets on a perkin-elmer 2000 at 8 cm À1 resolution in the region 4000-400 cm À1 . 1 H NMR spectra were carried out with bruker avance III HD 300/400 operating at 300/400 MHz using CDCl 3 solution with TMS at internal standard. 13 C NMR spectra obtained with bruker avance III HD 300 operating at 300 MHz using CDCl 3 solution with TMS at internal standard. Mass spectra recorded on LC-MS (ESI) mass spectrometer at 70 ev.

Procedure for antibacterial activity
The given compounds were screened for antibacterial activity against the bacteria bacillus licheniformis, bacillus species, escherichia coli, and staphylococcus aureus by using well diffusion method. The microbial suspension (100uL) having 108 CFU mL À1 of bacteria was carried out with the help of Mueller-Hinton agar (MHA) medium. The extracts were diluted by 100% dimethyl sulphoxide at the concentrations of 5 mg/mL and the given medium was melted and cooled to 48-50 C. The solid plates were formed by the addition of standardized inoculums (1.5 Â 108C FU/mL, 0.5McFarland) to the molten agar which was poured into sterile petri dishes. The well diffusion method was used to prepare product which was in the seeded agar plate; the compound activity had checked, and that was put into the well (6 mm). The plates were incubated in the incubator at 37 C for overnight. The antibacterial zone of inhibition of the extract had determined for the bacterial species in zone sizes around each well. The compounds produced diameters of the zone of inhibition as compared to standard ciprofloxacin [35,36].

Determination of minimum inhibitory concentration (MIC)
The broth dilution method is applicable for the minimum inhibitory concentration of given compounds. The concentration of compounds was prepared 8 mg/mL in the first tube containing 1mL of broth. The conduits were vortexes to make the initial standard concentration. These were serially diluted to other cannulas. Finally, 1mL compound was discarded from the last tube and prepared the dilution of 0.25, 0.50, 0.75, 1.0 mg/ mL. To all these tubes, 0.1mL of the log phase culture of target microorganisms were added separately and incubated at 37 C for 24-48 h for bacteria. After incubation, the lowest concentration of tube solution with no detectable bacterial growth was considered a minimum inhibitory concentration.

Antioxidant activity
2.2.4.1. Procedure for 2, 2-diphenyl-1-Picryl hydrazyl (DPPH) assay. DPPH (2, 2, diphenyl-1-picryl hydrazyl) radical scavenging assay was carried out [37] with slight modifications. The 1mL ethanolic solution had different concentrations of synthesized compounds. It was added with an equal volume of 0.1mm ethanolic solution of DPPH. The prepared solution settled for incubation at room temperature. The decreases in the concentration of DPPH were measured by noting the absorbance at 517nm. A similar test was performed with ascorbic acid, as an internal standard, instead of Schiff's base. The percentage scavenging of DPPH free radical for each concentration of test compounds had calculated the absorbance of negative control using Eq. (1).
Procedure for hydroxyl radical scavenging assay. Hydroxyl radical scavenging activities of given compounds were determined by using the earlier reported method [37]. The reaction cocktail included 60μL of 1mm FeCl 3 , 90μL of 1mm 1,10 phenanthroline, 2.4mL of 0.2M phosphate buffer (pH 7.8), 150μL of 0.17 M H 2 O 2 , and 1.5mL of various concentrations of each compound. The prepared solutions of given compounds were kept at room temperature for 5min incubation, and absorbance was measured at 560 nm with the help of spectrophotometer.
The α-tocopherol was used as the reference compound for hydroxyl radical scavenging assay.

Result and discussion
All the synthesized Schiff bases are in different colors and they are stable in air and moisture at room temperature. They are soluble in methanol, dimethyl sulphoxide, dimethylformamide, dichloromethane, chloroform, and partially soluble in ethanol. The spectral data of FT-IR, 1 H NMR, 13 C NMR, and LCMS confirmed their structure.

FT-IR analysis
FT-IR spectra of given compounds performed with the KBr pellet technique, and the observed results are in Table 2. The stretching bands are observed at 3444-3403 cm À1 due to the phenolic -OH group and 1665-1622 cm À1 due to the (-C¼N) stretching mode of the imine group.
The IR absorption peak at 1278-1263 cm À1 shows the phenolic ν(C-O) group with the presence of the keto-amine group having (N-O⋯H) intramolecular hydrogen bonding only in the solid-state [38]. The Schiff bases have shown two C-X stretching vibrations in the range of 794-760 cm À1 which confirms that the halogen groups are presented at the para position to the aromatic -OH group [38,39]. Even two stretching vibrations have been observed in the range 1573-1523 cm À1 and Table 2.  1413-1384 cm À1 due to the -NO 2 group. Table 2 and the supplementary file ( Fig. No. S1 to S11) represents spectroscopic data (IR) of given Schiff bases (3a-3k) (see Table 3).

1 H NMR analysis
1 H NMR spectral data of Schiff bases analyzed in CDCl 3 solvent, from the data, the presence of multiplets at δ value between 6.78-8.53ppm is due to aromatic protons. The appearance of the singlet at δ 13.92-14.09ppm shows the existence of the phenolic -OH proton having (N-O⋯H) intramolecular hydrogen bonding. In compounds (3a-3h) -CH 3 groups have appeared singlet at range δ 2.45-2.49ppm of 6H indicating that they are symmetrically equivalent. The compounds (3i, 3j, 3k), -CH 3 groups have shown two singlets at range δ 2.44-2.47ppm, which indicates that they are symmetrically non-equivalent. The spectral data of 1 H NMR is represented in the supplementary file (Fig. No.S12 to S22) of synthesized Schiff bases (3a-3k).

13 C NMR analysis
In 13 C NMR spectra, the peaks are observed at the range of δ value from 105 to 140ppm is due to aromatic and olefinic carbon. The signal present at δ 175.1-175.5ppm confirms azomethine (-C¼N-) group, and the phenolic C-OH carbon atom showed a sharp peak at δ 170.6-171.7ppm. The Schiff bases (3e, 3h, 3k) display signal at δ 165.1-164.5ppm due to a C-NO 2 bond. Schiff bases (3b-d, 3f, 3g, 3i, 3j) have exhibited a peak at δ 132.1-135.0ppm stipulates the presence of a C-X bond and the signal present at δ 14.3-14.6ppm due to -N¼C-CH 3 group. The spectral data of 13 C NMR is represented in the supplementary file (Fig. No. S23 to S33) of given Schiff bases (3a-3k).

LC-MS analysis
The LC-MS spectra of the Schiff bases exhibited different fragmentation patterns as expected, and results were getting to be in good agreement with their molecular formulae. -Cl and-Br containing Schiff bases gave different molecular ion peaks due to its isotopic effect. The molecular ion peak of the Schiff bases is displayed at [M þ H] þ peak. Especially the aromatic nitro Schiff bases (3e, 3h, 3k) lose -NO 2 radical and give molecular ion peak (M -NO 2 ) [39]. The spectral analysis (FT-IR, 1 H NMR, 13 C NMR, and LC-MS) confirmed that the synthesized compounds are Schiff bases. The mass spectrometry data of synthesized Schiff bases (3a-3k) is represented in the supplementary file (Fig. No.  S34 to S44).

Antibacterial activity
The experimental details and zone of inhibition of Schiff bases concerning the antibacterial activity against the bacterial strains is illustrated in table no.3. Among the eleven Schiff bases, the compound 3i has more potent against tested all four micro-organisms. The Schiff bases 3b, 3d, 3k are more effective against b. Lichenifermis and the compounds 3b, 3e, 3j show the higher activity against e. coli. Also, the compounds 3f, 3j are more potent against Bacillus sp., and compounds 3f, 3h give promising activity against s. aureus. The synthesized Schiff bases with the electron-withdrawing group such as [-Cl, -Br, -I, and -NO 2 ] and extended carbon chain of diamines suggest more potent in antibacterial activities. The impact of electronegative groups like -Cl,-Br, and -NO 2 is more effective than the -H and -I group on synthesized compounds. The antibacterial activity of these compounds is represented graphically in Figure 1.
The minimum inhibitory concentration was done at 0.25, 0.50, 0.75, and 1.0 mg/mL; the observed results are presented in Table 4. The Schiff base 3i has a good inhibition character at minimum concentration against referred microorganisms. It indicates the -Cl group and extended carbon chain of diamine which gives more potent biological activity. The Schiff bases 3b, 3d, 3k have shown good inhibition against b. licheniformis (gram-positive), and compounds 3f, 3h, 3k show high potent against b. species (gram-positive). The compounds 3e, 3j against e. coli (gram-negative) strain, and compounds 3f, 3h against s. aureus (grampositive) organisms have conveyed good inhibition at a minimum concentration (0.25 mg/mL).

Conclusion
The current study describes the synthesis of new eleven Schiff bases having an electronegative group (-Cl, -Br, -I, and -NO 2 ) located at para position to the phenolic -OH group. They are confirmed by using instrumental techniques viz. FT-IR, 1 H NMR, 13 C NMR, and LCMS analysis, also evaluated biologically by antibacterial and antioxidant activities. From the FT-IR analysis, we confirm the azomethine (-C¼N-) group, and the 1 H NMR peaks of the phenolic -OH proton are shown at δ 13.92-14.09ppm due to hydrogen bonding. The LC-MS analysis agrees with molecular ion peaks of synthesized Schiff bases. The screening data for antibacterial activity shows that electronegative-substituted Schiff bases are more potent against the tested gram-positive and gramnegative bacteria. Among these Schiff bases, compound 3i shows the best Antibacterial activity against all tested pathogens. From this, it is concluded that the influence of the -Cl group and extended carbon chain of diamines on synthesized compounds is more effective than other substituent's. The comparative determination of the total antioxidant capacity also shows the highest value for the electronegative Schiff bases with an extended carbon chain of diamines by DPPH and the hydroxyl radical scavenging method.

Author contribution statement
Bhagwat Vhanale: Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Digambar Kadam: Analyzed and interpreted the data.