Analar grade samples were used for the synthesis of Schiff bases. 5, 5-dimethylcyclohexanone, 2-aminophenol, and aniline, were purchased from E. Merck. The percentage of elements such as carbon, hydrogen, and nitrogen were analysed by microanalysis using Elementar make Vario EL III model CHNS analyser. KBr disc technique on a Shimadzu model FT-IR Spectrometer (Model IR affinity) were used for recording IR spectra in the region 4000–400 cm−1. Shimadzu UV-Visible-1800 Spectrophotometer was used for recording electronic spectra in DMSO. BRUKER AVANCE III HD was used for 1H NMR and 13C NMR studies in dmso-d6. Mass spectra were recorded using QP 2010 model Shimadzu GCMS.
Synthesis and characterization of Schiff bases
To a stirred ethanolic solution of 2-aminophenol (0 .02 mol), 5,5-dimethyl-1,3-cyclohexanedione (0.01 mol) dissolved in hot ethanol was added, refluxed for 20 min and cooled. Brown-coloured precipitate formed was filtered, washed with ethanol, and recrystallized. Yield was 80%, and M.P. 180 °C .
Anal.calcd for C20H22N2O2 was C, 74.53; H, 6.83; and N, 8.69%. Found. was C, 73.91; H, 6.74; and N, 8.52%; IR (KBr) was 3240 cm−1 (OH), 1600 cm−1 (C=N), 1238 cm−1 (C-O), 3080 cm−1 (aromatic C-H), and 2960 and 2877 cm−1 (aliphatic C-H); UV was 22936 cm−1 (n → π*), and 33784 and 39682 cm−1 (π → π*); 1H NMR was δ 0.99 (CH3), δ 2.35 (CH2 between two azomethine group), δ 2.02 (CH2 adjacent to > C(CH3)2), and δ 6.81–7.07 (aromatic H); 13C NMR was 95.88ppm (C=N), 27.84ppm (CH3), 41.52ppm (C containing CH3), 49.78ppm (C between azomethine groups), 32.32ppm (C adjacent to > C(CH3)2), and 116.28–151.55 ppm (aromatic C); mass was M+ peak absent, m/z 216 (base peak) [C14H18NO]+, m/z 231 [C14H19N2O]+, and m/z 178 [C11H16NO]+.
N, N’-(5,5-dimethylcyclohexane-1,3-diylidene)dianiline (DmChDa)
0.01 mol of 5,5-dimethyl-1,3-cyclohexanedione was dissolved in hot ethanol and added to a stirred ethanolic solution of 0.02 mol of aniline. Refluxed for 3h and cooled. Yellow precipitate separated was filtered, washed with ethanol, and recrystallized. Yield was 78%, and M.P. 152 °C .
Anal.calcd for C20H22N2 was C, 82.7; H, 7.5; and N, 9.6%. Found. was C, 81.3; H, 6.9; and N, 8.9%; IR (KBr) was 1564 cm−1 (C=N); 1249 and 3234 cm−1 (N-H); 2949, 2879, and 2810 cm−1 (CH3 and CH2); and 3055 cm−1 (aromatic H); UV was 32020 cm−1 (n → π*), and 39463 cm−1 (π → π*); 1H NMR was δ 1.04 (CH3), δ 1.51 (CH2 between two azomethine group), and δ 7.26,7.09 (aromatic H); 13C NMR was 28.35 ppm (CH3); 98.94 ppm (C=N); 32.88, 43.71, and 50.35 ppm (two CH2); and 123.39–138.14 ppm (aromatic C); mass was M+ peak absent, m/z 159 (base peak-[C11H13N]+), m/z 215 [C14H20N2]+, and m/z 198 [C14H18N]+.
In vitro antibacterial studies
Mueller-Hinton agar was used for preparing the medium . Schiff base compounds and the standard antibiotic ampicillin were dissolved in DMSO to prepare the stock solutions. Then, it is diluted to obtain various ranges of concentrations from 100 to 500 μg disc−1. Disc diffusion method was adopted for the drug . The petri dishes were incubated in an air ambiance at 35 °C for 24 h. The diameter of the zone of inhibition was measured and compared with zones produced by the reference antibiotic, ampicillin.
Target proteins in Staphylococcus aureus
Staphylococcus aureus sortase-A (PDB ID: 1T2P)
Sortases are extracellular transpeptidases of Gram-positive bacteria [23, 24]. The function of the enzyme is to sort proteins into the cell wall compartment of Gram-positive bacteria, hence named Sortases. Sortases have a great role in the cell wall envelope assembly and bacterial pathogenicity.
DNA gyrase (PDB ID: 3U2D)
Topoisomerase is an isomerase enzyme that provokes dramatic change in the topology of DNA structure [25, 26]. Topoisomerase is categorized as topoisomerase I and topoisomerase II, based on the number of strands cut in one phase of action. New topoisomerases, type III and IV have also been discovered recently. DNA gyrase subclass of type II topoisomerase is responsible primarily for DNA replication.
Dihydrofolate reductase (DHFR) (PDB ID: 2W9S)
Dihydrofolate reductase (DHFR) is an enzyme that catalyses the formation of tetrahydrofolate (THF) by the reduction of Dihydrofolate (DHF) in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) [27, 28]. Also, it has a great role in the synthesis of thymidylate, purines, methionine, and some other important metabolites. These enzymes are required for cell proliferation. Thus, inhibition of dihydrofolate reductase will results in the destruction of the intracellular tetrahydrofolate pool thereby preventing biosynthesis of RNA, DNA, thymidine, and protein. Due to the wide range of cellular functions, they are targets for anticancer and antimicrobial agents.
Clumping factor A (ClfA) (PDB ID: 1N67)
In blood plasma, there is a glycoprotein called fibrinogen (Fg) which is present at ~ 3 mg/ml concentration and has a significant role in coagulation and haemostasis. Six polypeptide chains are present in fibrinogen such as 2 Aa, 2 Bb, and 2 nd 2 Bb, which are dimeric and symmetrical. The γ-chain has C-terminal residues which are biologically important. In the process of fibrinogen-dependent platelet adherence and aggregation, they interact with platelet integrin aIIb3. This C-terminal residue of γ-chain is also targeted by the pathogenic bacterium Staphylococcus aureus, resulting in fibrinogen-dependent cell clumping and tissue adherence. Clumping factor A (ClfA) [29, 30] was the first Fg γ-chain-binding S. aureus adhesin identified.
Dehydrosqualene synthase (CrtM) (PDB ID: 2ZCO)
The golden carotenoid pigment staphyloxanthin is a virulence factor for S. aureus. Dehydrosqualene synthase [31, 32] is involved in the synthesis of this pigment. The main responsibility of the pigment is to preserve S. aureus against oxidative stress as a result of host immune defence by reactive oxygen species and neutrophils by acting as an antioxidant.
Undecaprenyl diphosphate synthase (UPPS) (PDB ID: 4H8E)
The role of undecaprenyl diphosphate synthase (UPPS) in the biosynthesis of the cell wall of Staphylococcus aureus is very significant [33, 34]. UPPS is important since it is vital for the formation of peptidoglycan. Also, UPPS is not present in humans and is additional merit for the development of good antibacterial agents.
In silico molecular docking studies
Lipinski rule of five
Lipinski rule envisages that an orally active drug will be small and slightly lipophilic. This rule depicts molecular properties rather than pharmacological activity and states that a drug has good oral activity if it satisfies the five conditions such as the following :
Molecular weight < 500
Octanol-water partition coefficient logP < 5
Less than 5 hydrogen bond donors (total number of NH and OH bonds)
Less than 10 hydrogen bond acceptors (total number of N and O atoms)
Molar refractivity between 40 and 130
Docking studies were carried out to establish the mechanism by which the Schiff base compounds prevent bacterial growth. Binding affinity and interactions of the Schiff bases with different target proteins in S. aureus were derived from docking studies [36,37,38]. The steps involved in the docking process are as follows.
Preparation of ligands and proteins
The structure of the Schiff bases in MOL format was derived using ChemSketch software and converted to PDB format using open babel software. The structures of the proteins were downloaded in PDB format from RCSB PDB. Using Pymol software, water molecules and ligands already present in the proteins were removed; hydrogen atoms were added and saved in PDB format. Six target proteins of Staphylococcus aureus were utilized to check the interaction with synthesized Schiff bases.
Prediction of active site
Prediction of the active site is important in structure-based drug design. Co-ordinates of binding sites of the proteins were identified using the software BIOVIA Discovery Studio.
Molecular docking calculations were carried out with the aid of the software AutoDock 4.2 and binding energy of the protein—Schiff base adducts were obtained .
Visualization of protein-ligand complexes
The protein-ligand complexes were visualized using the software BIOVIA Discovery Studio and their 3D and 2D interaction plots were derived. Hydrogen bond interactions such as conventional and non-conventional H bonds, hydrophobic interactions such as amide-pi stacked, pi-pi stacked, pi-sigma, pi-pi T-shaped, alkyl and pi-alkyl interactions, electrostatic interactions such as pi-anion and pi-cation interactions, van der Waals interaction, and unfavourable donor-donor and acceptor-acceptor interactions are commonly seen between protein and ligand. The binding affinity of the compound with the target protein is the resultant of all the interactions and binding energy existing between them.